EA045730B1 - ANTI-MUC16 (MUCIN16) ANTIBODIES - Google Patents

Description

Link to sequence list

This application incorporates by reference a sequence listing provided in machine-readable form as file 10295WO02-Sequence.txt, created on September 22, 2017, containing 893,983 bytes. 1

Technical field

This invention relates to antibodies, and antigen-binding fragments thereof, that are specific for mucin 16 (MUC16), and methods of using them. The present invention also relates to bispecific antigen binding molecules that bind MUC16 and CD3, and methods of using them. This invention also provides antibody-drug conjugates comprising an anti-MUC16 antibody or fragment thereof and a therapeutic agent (eg, a cytotoxic agent).

State of the art

Mucin 16 (MUC16), also known as cancer antigen 125, carcinoma antigen 125, carbohydrate antigen 125, or CA-125, is a highly glycosylated integral membrane glycoprotein with a single transmembrane domain that is expressed at high levels in ovarian cancer. MUC16 consists of three main domains: an extracellular N-terminal domain, a large tandem repeat domain that is interrupted by sea urchin sperm protein, enterokinase, and agrin (SEA) domains, and a carboxyl-terminal domain that contains a transmembrane segment and a short cytoplasmic tail. Proteolytic cleavage results in the release of most of the extracellular portion of MUC16 into the bloodstream. MUC16 is overexpressed in cancers, including ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, mass-forming intrahepatic cholangiocarcinoma, cervical adenocarcinoma and gastric adenocarcinoma, as well as in diseases and conditions including inflammatory bowel disease, liver cirrhosis , heart failure, peritoneal infection and abdominal surgery. (Haridas, D. et al., 2014, FASEB J., 28: 4183-4199). Expression on cancer cells has been shown to protect tumor cells from the immune system. (Felder, M. et al., 2014, Molecular Cancer, 13: 129). Treatments for ovarian cancer using antibodies to MUC16 have been investigated. Oregovomab and abgovomab are anti-MUC16 antibodies that have had limited success. (Felder, supra, Das, S. and Batra, SK 2015, Cancer Res. 75: 4660-4674.)

CD3 is a homodimeric or heterodimeric antigen expressed on T cells in association with the T cell receptor (TCR) complex and is required for T cell activation. Functional CD3 is formed from the dimeric association of two of four different chains: epsilon, zeta, delta and gamma. CD3 dimer patterns include gamma/epsilon, delta/epsilon, and zeta/zeta. Anti-CD3 antibodies have been shown to cluster CD3 on T cells, thereby causing T cell activation in a manner similar to TCR engagement by peptide-loaded MHC molecules. Thus, anti-CD3 antibodies have been proposed for therapeutic purposes involving T cell activation. In addition, bispecific antibodies that are capable of binding CD3 and a target antigen have been proposed for therapeutic applications involving targeting T cell immune responses to tissues and cells expressing the target antigen.

Antigen-binding molecules that target MUC16, including antibody-drug conjugates, as well as bispecific antigen-binding molecules that bind to both MUC16 and CD3, will be useful in therapeutic settings in which specific targeting and T cell killing of cells that express MUC16 is desirable.

Brief description of the invention

In a first aspect, the present invention relates to antibodies and antigen-binding fragments thereof that bind to human MUC16. Antibodies according to this aspect of the invention are useful in particular for targeting cells expressing MUC16. The present invention also relates to bispecific antibodies and antigen binding fragments thereof that bind human MUC16 and human CD3. Bispecific antibodies in accordance with this aspect of the invention are useful, in particular, for targeting T cells expressing CD3 and for promoting T cell activation, for example, in conditions where T cell-mediated killing of MUC16 expressing cells is useful or desirable. For example, bispecific antibodies can direct CD3-mediated T cell activation to specific MUC16-expressing cells, such as ovarian tumor cells.

Exemplary anti-MUC16 antibodies of the present invention are listed in table. 1 and 2 of this document. In table 1 shows identifiers of the amino acid sequences of the heavy chain variable regions (HCVR) and the light chain variable regions (LCVR), as well as the regions that determine the complementarity of the heavy chain (HCDR1, HCDR2 and HCDR3) and the regions that determine the complementarity of the light chain (LCDR1, LCDR2 and LCDR3) typical anti-MUC16 antibodies. In table 2 shows identifiers of the sequences of nucleic acid molecules encoding HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of typical anti-MUC16 antibodies.

This invention relates to antibodies or antigen-binding fragments thereof containing

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HCVR containing an amino acid sequence selected from any of the HCVR amino acid sequences listed in table. 1, or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

This invention also relates to antibodies or antigen-binding fragments thereof containing LCVR containing an amino acid sequence selected from any of the LCVR amino acid sequences listed in table. 1, or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

This invention also relates to antibodies or antigen-binding fragments thereof containing a paired amino acid sequence of HCVR and LCVR (HCVR/LCVR), containing any of the amino acid sequences of HCVR listed in table. 1, paired with any of the LCVR amino acid sequences listed in table. 1. In accordance with some embodiments, the present invention provides antibodies or antigen binding fragments thereof comprising the paired amino acid sequence HCVR/LCVR contained in any of the exemplary anti-MUC16 antibodies listed in Table. 1. In some embodiments, the amino acid sequence pair HCVR/LCVR has the sequence SEQ ID NO: 18/26 (eg, H1H8767P).

This invention also relates to antibodies or antigen-binding fragments thereof comprising a heavy chain CDR1 (HCDR1) containing an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in table. 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments thereof comprising a heavy chain CDR2 (HCDR2) containing an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in table. 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

This invention also relates to antibodies or antigen-binding fragments thereof containing a heavy chain CDR3 (HCDR3) containing an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in table. 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigen binding fragments thereof comprising a light chain CDR1 (LCDR1) containing an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table. 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

This invention also relates to antibodies or antigen-binding fragments thereof comprising a light chain CDR2 (LCDR2) containing an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in table. 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

This invention also relates to antibodies or antigen-binding fragments thereof containing a light chain CDR3 (LCDR3) containing an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in table. 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

This invention also relates to antibodies or antigen-binding fragments thereof containing a paired amino acid sequence of HCDR3 and LCDR3 (HCDR3/LCDR3), containing any of the amino acid sequences of HCDR3 listed in table. 1, paired with any of the LCDR3 amino acid sequences listed in table. 1. In accordance with some embodiments, the present invention provides antibodies or antigen binding fragments thereof comprising the paired amino acid sequence HCDR3/LCDR3 contained in any of the exemplary anti-MUC16 antibodies listed in Table. 1.

In some embodiments, the HCDR3/LCDR3 amino acid sequence pair has the sequence SEQ ID NO: 24/32 (eg, H1H8767P).

The present invention also provides antibodies or antigen binding fragments thereof comprising a set of six CDRs (ie, HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-MUC16 antibodies listed in Table. 1. In some embodiments of the invention, the set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2LCDR3 is selected from the group consisting of SEQ ID NO: 20-22-24-28-30-32 (for example, H1H8767P).

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In a similar embodiment, the present invention provides antibodies or antigen-binding fragments thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1LCDR2-LCDR3) contained in the paired amino acid sequence HCVR/LCVR, as defined by any of the generic anti-MUC16 antibodies listed in table. 1. For example, this invention includes antibodies or antigen-binding fragments thereof containing the set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 contained in the pair of amino acid sequences HCVR/LCVR, sequence SEQ ID NO: 18/26 (for example, H1H8767P ). Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in said HCVR and/or LCVR amino acid sequences described herein. Typical conventions that can be applied to define the boundaries of the CDR include, for example, the Kabat definition, the Chotia definition and the AtM definition. In general, the Kabat definition is based on sequence variability, the Chotiya definition is based on the location of structural loop regions, and the AtM definition is a compromise between the Kabat and Chotiya approaches; see, for example, Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD. (1991); Al-Lazikani et al., J. Mol. Biol. 273:921-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9212 (1989). Public databases are also available to identify CDR sequences in an antibody.

The present invention also relates to nucleic acid molecules encoding anti-MUC16 antibodies or portions thereof. For example, this invention relates to nucleic acid molecules encoding any of the HCVR amino acid sequences listed in table. 1; in certain embodiments of the present invention, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

This invention also relates to nucleic acid molecules encoding any of the LCVR amino acid sequences listed in table. 1; in certain embodiments of the present invention, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

This invention also relates to nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in table. 1; in certain embodiments of the present invention, the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in table. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

This invention also relates to nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in table. 1; in certain embodiments of the present invention, the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in table. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

This invention also relates to nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in table. 1; in certain embodiments of the present invention, the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in table. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

This invention also relates to nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in table. 1; in certain embodiments of the present invention, the nucleic acid molecule contains a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in table. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

This invention also relates to nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in table. 1; in certain embodiments of the present invention, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

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This invention also relates to nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in table. 1; in certain embodiments of the present invention, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

This invention also provides nucleic acid molecules encoding HCVR, wherein HCVR contains a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the set of amino acid sequences HCDR1-HCDR2-HCDR3 is as defined by any generic anti-MUC16 antibodies listed in table. 1.

This invention also provides nucleic acid molecules encoding LCVR, wherein LCVR contains a set of three CDRs (i.e. LCDR1-LCDR2-LCDR3), wherein the set of amino acid sequences LCDR1-LCDR2-LCDR3 is as defined by any generic anti-MUC16 antibodies listed in table. 1.

This invention also provides nucleic acid molecules encoding both HCVR and LCVR, wherein HCVR contains the amino acid sequence of any of the HCVR amino acid sequences listed in table. 1, and wherein the LCVR contains the amino acid sequence of any of the LCVR amino acid sequences listed in table. 1. In some embodiments of the present invention, the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in table. 2, or a substantially similar sequence thereto, having a sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% identity and a polynucleotide sequence selected from any nucleic acid sequence LCVR listed in table. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some embodiments of the present invention, in accordance with this aspect of the invention, the nucleic acid molecule encodes HCVR and LCVR, wherein HCVR and LCVR are both derived from the same anti-MUC16 antibody specified in table. 1.

The present invention also provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-MUC16 antibody. For example, the present invention includes recombinant expression vectors containing any of the nucleic acid molecules mentioned above, that is, nucleic acid molecules encoding any of the HCVR, LCVR and/or CDR sequences as set forth in Table. 1. Also included within the scope of this invention are the host cells into which such vectors are introduced, as well as methods for producing antibodies or parts thereof by culturing the host cells under conditions allowing the production of antibodies or antibody fragments, and recovering the antibodies and antibody fragments so obtained way.

This invention includes anti-MUC16 antibodies having a modified glycosylation profile. In some embodiments of the present invention, modification to remove unwanted glycosylation sites or an antibody lacking a fucose moiety present in the oligosaccharide chain may be useful, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277 :26733). In other applications, modification of galactosylation can be made to alter complement-dependent cytotoxicity (CDC).

In another aspect, the present invention provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds MUC16 and a pharmaceutically acceptable carrier. In a related aspect, the present invention provides a composition that is a combination of an anti-MUC16 antibody and a second therapeutic agent. In one embodiment of the present invention, the second therapeutic agent is any agent that preferentially binds to an anti-MUC16 antibody. Additional combination therapies and combination formulations comprising the anti-MUC16 antibodies of this invention are described elsewhere herein.

In another aspect, the present invention provides therapeutic methods for targeting/killing tumor cells expressing MUC16 using an anti-MUC16 antibody of the present invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an anti-MUC16 antibody of the present invention to a subject who needs it. In some cases, anti-MUC16 antibodies (or antigen-binding fragments thereof) may be used to treat cancer (eg, ovarian cancer) or may be modified to be more cytotoxic by methods including, but not limited to, modified Fc domains to increase ADCC (see, for example, Shield et al. (2002) JBC 277: 26733), radioimmunotherapy, antibody-drug conjugates, or other methods for increasing the efficiency of tumor ablation.

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The present invention also includes the use of an anti-MiC16 antibody of the present invention in the manufacture of a medicament for the treatment of a disease or disorder (eg, cancer) associated with or caused by MUC16-expressing cells. In one aspect, the present invention provides a compound containing an anti-MUC16 antibody or an antigen-binding fragment thereof, or a MUC16xCD3 bispecific antibody as described herein, for use in medicine. In one aspect, the present invention relates to a compound containing an antibody-drug conjugate (ADC), as described herein, for use in medicine.

In yet another aspect, the present invention relates to monospecific anti-MUC16 antibodies for diagnostic applications, such as, for example, imaging reagents.

In yet another aspect, the present invention provides therapeutic methods for stimulating T cell activation using an anti-CD3 antibody or an antigen binding portion of an antibody of the present invention, the therapeutic methods comprising administering a therapeutically effective amount of a pharmaceutical composition comprising the antibody.

In another aspect, the present invention provides an isolated antibody or antigen binding fragment thereof that binds human mucin 16 (MUC16) with a binding dissociation equilibrium constant (KD) of less than about 53 nM, as measured by surface plasmon resonance assay at 25°C. In yet another aspect, the present invention provides an isolated antibody or antigen binding fragment thereof that binds human MUC16 with a dissociative half-life (t1/ ₂ ) greater than about 15 minutes, as measured in a surface plasmon resonance assay at 25°C.

The present invention also provides an antibody or antigen binding fragment that competes for binding to human MUC16 with a reference antibody containing the amino acid sequence pair HCVR/LCVR as set forth in Table. 1. In another aspect, the present invention provides an antibody or antigen binding fragment that competes for binding to human MUC16 with a reference antibody comprising the amino acid sequence pair HCVR/LCVR selected from the group consisting of SEQ iD NO: 2/10; 18/26; 34/42; 50/58, 66/74; 82/90; 98/106; 114/122; 130/138; 146/154; 162/170; 178/186; 194/394; 202/210; 218/226, 234/242; 250/1936; 258/266; 274/1936; 282/290; 298/306; 314/322; 330/338; 346/354; 362/370; and 378/386.

The invention further provides an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment thereof binds to the same epitope on human MUC16 as the reference antibody comprising the amino acid sequence pair HCVR/LCVR as set forth in Table 1. 1. In another aspect, the antibody or antigen binding fragment thereof binds to the same epitope on human MUC16 as the reference antibody comprising the amino acid sequence pair HCVR/LCVR selected from the group consisting of SEQ ID NO: 2/10; 18/26; 34/42; 50/58, 66/74; 82/90; 98/106; 114/122; 130/138; 146/154; 162/170; 178/186; 194/394; 202/210; 218/226, 234/242; 250/1936; 258/266; 274/1936; 282/290; 298/306; 314/322; 330/338; 346/354; 362/370; and 378/386.

This invention also provides an isolated antibody or antigen-binding fragment thereof that binds human MUC16, wherein the antibody or antigen-binding fragment comprises: heavy chain variable region (HCVR) complementarity determining regions (CDRs) having the amino acid sequence set forth in Table. 1; and a light chain variable region (LCVR) CDR having the amino acid sequence as shown in table. 1. In another aspect, the isolated antibody or antigen binding fragment comprises a heavy and light chain CDR of a pair of amino acid sequences HCVR/LCVR selected from the group consisting of: 2/10; 18/26; 34/42; 50/58, 66/74; 82/90; 98/106; 114/122; 130/138; 146/154; 162/170; 178/186; 194/394; 202/210; 218/226, 234/242; 250/1936; 258/266; 274/1936; 282/290; 298/306; 314/322; 330/338; 346/354; 362/370; and 378/386. In yet another aspect, the isolated antibody or antigen binding fragment thereof comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, selected from the group consisting of: SEQ ID NO: 4-6-8-12-14-16; 20-22-24-28-30-32; 36-38-40-44-46-48; 52-54-56-60-62-64; 68-70-72-76-78-80; 84-86-88-92-94-96; 100-102-104-108-110-112; 116-118-120-124-12 6-128; 132-134-13 6140-142-144; 148-150-152-156-158-160; 164-166-168-172-174-176; 180-182-184-188-190-192; 196-198-200396-398-400; 204-206-208-212-214-216; 220-222-224-228-230-232; 236-238-240-244-246-248; 252-254-2561938-1940-1942; 260-262-264-268-270-272; 276-278-280-1938-1940-1942; 284-286-288-292-294-296; 300302-304-308-310-312; 316-318-320-324-326-328; 332-334-336-340-342-344; 348-350-352-356-358-360; 364366-368-372-374-376; and 380-382-384-388-390-392.

In another aspect, the present invention provides an isolated antibody or antigen binding fragment thereof that binds human MUC16, wherein the antibody or antigen binding fragment comprises: (a) a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO : 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 202, 218, 234, 250, 258, 274, 282, 298, 314, 330, 346, 362 and 378; and (b) a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 10; 26; 42; 58, 74; 90; 106; 122; 138; 154; 170; 186; 210; 226, 242; 266; 290; 306; 322; 338; 354; 370; 386; 1936 and 394. In a further aspect, the isolated antibody or antigen-binding

- 5 045730 fragment according to claim 10, in which the antibody or antigen binding fragment contains a pair of amino acid sequences HCVR/LCVR selected from the group consisting of: SEQ ID NO: 2/10; 18/26; 34/42; 50/58, 66/74; 82/90; 98/106; 114/122; 130/138; 146/154; 162/170; 178/186; 194/394; 202/210; 218/226, 234/242; 250/1936; 258/266; 274/1936; 282/290; 298/306; 314/322; 330/338; 346/354; 362/370 and 378/386.

This invention also provides an isolated antibody or antigen binding fragment thereof that binds to human MUC16 at the epitope ranging from residue 428 to residue 481 of SEQ ID NO: 1902. In some cases, the isolated antibody or antigen binding fragment thereof interacts with amino acid residues 428-434, 429-434, 453-467, 459-467, 460-467 and/or 474481 SEQ ID NO: 1902. In some embodiments, the antibody or antigen binding fragment thereof reacts with amino acid residues 428-434, 429-434, 453- 467, 459-467, 460-467 and 474-481 SEQ ID NO: 1902. This invention further provides an isolated antibody or antigen binding fragment thereof that binds to human MUC16 at the epitope ranging from residue 126 to residue 138 of SEQ ID NO: 1902. In some cases, the isolated antibody or antigen binding fragment thereof reacts with amino acid residues 126-131, 127-131 and/or 132-138 of SEQ ID NO: 1902. In some embodiments, the antibody or antigen binding fragment thereof reacts with amino acid residues 12 6-131, 127-131 and 132138 SEQ ID NO: 1902. This invention further provides an isolated antibody or antigen binding fragment thereof that binds to human MUC16 at the epitope ranging from residue 357 to residue 369 of SEQ ID NO: 1902 In some cases, the isolated antibody or antigen binding fragment thereof reacts with amino acid residues 357-369, 358-366, 358-369 and/or 361369 of SEQ ID NO: 1902. In some embodiments, the antibody or antigen binding fragment thereof reacts with amino acid residues 357-369, 358-366, 358-369 and 361369 SEQ ID NO: 1902. This invention further provides an isolated antibody or antigen binding fragment thereof that binds human MUC16 at one or more of the five membrane-proximal SEA domains Human MUC 16 (SEQ ID NO: 1899). The five membrane-proximal SEA domains correspond to residues 13791-14451 of SEQ ID NO: 1899. In some cases, an antibody or antigen binding fragment thereof binds to a K _D of less than about 60 nM, as measured in a surface plasmon resonance assay at 25°C. In some embodiments, the antibody or antigen binding fragment thereof binds at residues 14237 to 14290 of SEQ ID NO: 1899. In one embodiment, the antibody or antigen binding fragment thereof comprises a CDR of an HCVR/LCVR pair comprising the amino acid sequences of SEQ ID NO: 18 /26. In some embodiments, the antibody or antigen binding fragment thereof binds at residues 13935 to 13947 of SEQ ID NO: 1899. In one embodiment, the antibody or antigen binding fragment thereof comprises a CDR of an HCVR/LCVR pair comprising the amino acid sequences of SEQ ID NO: 82 /858. In some embodiments, the antibody or antigen binding fragment thereof binds at residues 14165 to 14178 of SEQ ID NO: 1899. In one embodiment, the antibody or antigen binding fragment thereof comprises a CDR of an HCVR/LCVR pair comprising the amino acid sequences of SEQ ID NO: 98 /170.

In one aspect, the present invention provides antibodies or antigen-binding fragments thereof that bind to one of several SEA domains of MUC16. In various embodiments of the invention, anti-MUC16 antibodies or antigen binding fragments bind to any one of SEA1, SEA2, SEA3, SEA4, SEA5, SEA6, SEA7, SEA8, SEA9, SEA10, SEA11, SEA12, SEA13, SEA14, SEA15, or SEA16. In one embodiment of the invention, the anti-MUC16 antibody or fragment thereof binds to SEA1 (residues 12074 to 12229 of SEQ ID NO: 1899). In one embodiment of the invention, the anti-MUC16 antibody or fragment thereof binds to SEA2 (residues 12230 to 12387 of SEQ ID NO: 1899). In one embodiment of the invention, the anti-MUC16 antibody or fragment thereof binds to SEA3 (residues 12388 to 12543 of SEQ ID NO: 1899). In one embodiment of the invention, the anti-MUC16 antibody or fragment thereof binds to SEA4 (residues 12544 to 12698 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody or fragment thereof binds to SEA5 (residues 12699 to 12854 of SEQ ID NO: 1899). In one embodiment of the invention, the anti-MUC16 antibody or fragment thereof binds to SEA6 (residues 12855 to 13010 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody or fragment thereof binds to SEA7 (residues 13011 to 13166 of SEQ ID NO: 1899). In one embodiment of the invention, the anti-MUC16 antibody or fragment thereof binds to SEA8 (residues 13167 to 13323 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody or fragment thereof binds to SEA9 (residues 13324 to 13478 of SEQ ID NO: 1899). In one embodiment of the invention, the anti-MUC16 antibody or fragment thereof binds to SEA10 (residues 13479 to 13634 of SEQ ID NO: 1899). In one embodiment of the invention, the anti-MUC16 antibody or fragment thereof binds to SEA11 (residues 13635 to 13790 of SEQ ID NO: 1899). In one embodiment of the invention, the anti-MUC16 antibody or fragment thereof binds to SEA12 (residues 13791 to 13923 of SEQ ID NO: 1899). In one embodiment of the invention, anti-MUC16

- 6 045730 antibody or fragment thereof binds to SEA13 (residues 13924 to 14074 of SEQ ID NO: 1899). In one embodiment of the invention, the anti-MUC16 antibody or fragment thereof binds to SEA14 (residues 14075 to 14227 of SEQ ID NO: 1899). In one embodiment of the invention, the α-anti-MUC16 antibody or fragment thereof binds to SEA15 (residues 14228 to 14320 of SEQ ID NO: 1899). In one embodiment of the invention, the anti-MUC16 antibody or fragment thereof binds to SEA16 (residues 14321 to 14464 of SEQ ID NO: 1899).

In accordance with another aspect, the present invention relates to antibody-drug conjugates comprising an anti-MUC16 antibody or antigen-binding moiety and a therapeutic agent (eg, a cytotoxic agent). In some embodiments, the antibody or antigen-binding fragment thereof and the cytotoxic agent are covalently linked through a linker, as discussed herein. In various embodiments of the invention, the anti-MUC16 antibody or antigen binding fragment thereof can be any of the anti-MUC16 antibodies or fragments described herein.

In some embodiments, the cytotoxic agent is selected from auristatin, a maytansinoid, tubulisin, a tomaimycin derivative, or a dolastatin derivative. In some cases, the cytotoxic agent is an auristatin selected from MMAE or MMAF, or a maytansinoid selected from DM1 or DM4. In some embodiments, the cytotoxic agent is a maytansinoid having the structure of formula (I) or formula (II), as discussed herein.

In some embodiments of the invention, the cytotoxic agent is a maytansinoid having the structure:

o sn ₃

In some embodiments of the invention, the cytotoxic agent is a maytansinoid having the structure:

o sn ₃

In some embodiments, the antibody-drug conjugate comprises an anti-MUC16 antibody or fragment thereof, and

Where “ represents a linkage to an anti-MUC16 antibody or fragment thereof.

In some embodiments, the antibody-drug conjugate comprises an anti-MUC16 antibody or fragment thereof, and

- 7 045730 where “ represents a connection with an anti-MiC16 antibody or a fragment thereof.

In some embodiments, the antibody-drug conjugate comprises an anti-MUC16 antibody or fragment thereof, and

where < represents a linkage to an anti-MUC16 antibody or fragment thereof.

In some embodiments, the bond contacts the antibody or fragment thereof through the sulfur moiety of the cysteine residue.

In some embodiments, the antibody-drug conjugate comprises an anti-MUC16 antibody or fragment thereof, and

o or

o or a mixture thereof, where g represents a linkage to an anti-MUC16 antibody or a fragment thereof.

In some embodiments, the bond contacts the antibody or fragment thereof through the nitrogen moiety of the lysine residue.

In any of the various embodiments of the antibody-drug conjugates described above or herein, the antibody-drug conjugate may contain from 1 to 4 cytotoxic agents per anti-MUC16 antibody or fragment thereof.

In accordance with another aspect, the present invention provides bispecific antigen-binding molecules (eg, antibodies) that bind to MUC16 and CD3. Such bispecific antigen binding molecules are also referred to herein as anti-MUC16/anti-CD3 bispecific molecules, anti-CD3/anti-MUC16 bispecific molecules, or MUC16xCD3 bsAbs. The anti-MUC16 portion of the anti-MUC16/anti-CD3 bispecific molecule is useful for targeting cells (eg, tumor cells) that express MUC16 (eg, ovarian tumors), and the anti-CD3 portion of the bispecific molecule is useful for activating T cells. Simultaneous binding to MUC16 on the tumor cell and CD3 on the T cell facilitates targeted killing (cell lysis) of the target tumor cell activated by the T cell. Therefore, the anti-MUC16/anti-CD3 bispecific molecules of the present invention are therefore useful, among other things, for the treatment of diseases and disorders associated with or caused by MUC16-expressing tumors (eg, ovarian cancer).

Bispecific antigen binding molecules in accordance with this aspect of the invention comprise a first antigen binding domain that specifically binds to human CD3 and a second antigen binding domain that specifically binds to MUC16. This invention includes anti-MUC16/αanti-CD3 bispecific molecules (e.g., bispecific antibodies), in

- 8 045730 wherein each antigen binding domain contains a heavy chain variable region (HCVR) paired with a light chain variable region (LCVR). In some illustrative embodiments of the invention, the anti-CD3 antigen binding domain and the anti-MUC16 antigen binding domain each contain different, distinct HCVRs paired with a common LCVR. For example, as shown in Example 3 herein, bispecific antibodies have been constructed comprising a first antigen binding domain that specifically binds CD3, wherein the first antigen binding domain comprises an HCVR derived from an anti-CD3 antibody coupled to an LCVR derived from an anti-MUC16 antibodies (eg, the same LCVR that is included in the antiMUC16 antigen binding domain); and a second antigen binding domain that specifically binds to MUC16, the second antigen binding domain comprising HCVR/LCVR derived from an anti-MUC16 antibody. In other words, in the exemplary molecules described herein, pairing of HCVR from an anti-CD3 antibody with LCVR from an anti-MUC16 antibody creates an antigen binding domain that specifically binds to CD3 (but does not bind to MUC16). In such embodiments, the first and second antigen binding domains contain different anti-CD3 and anti-MUC16 HCVRs, but share a common anti-MUC16 LCVR. In other embodiments, the bispecific antigen binding molecules contain different anti-CD3 and anti-MUC16 HCVRs, but have a common LCVR. Amino acid sequence of this LCVR. is shown, for example, in SEQ ID NO: 18-90, and the amino acid sequences of the corresponding CDRs (ie LCDR1-LCDR2-LCDR3) are shown in SEQ ID NO: 1892, 1894 and 1896, respectively. Genetically modified mice can be used to produce fully human bispecific antigen binding molecules containing two different heavy chains that are linked to an identical light chain that contains a variable domain derived from one of two different human light chain variable region gene segments. Alternatively, the variable heavy chains can be paired with a single common light chain and expressed recombinantly in host cells. Thus, the antibodies of this invention may contain immunoglobulin heavy chains linked to a single rearranged light chain. In some embodiments, the light chain comprises a variable domain derived from a human Vk1-39 gene segment or a Vk3-20 gene segment. In other embodiments, the light chain comprises a variable domain derived from a human Vk1-39 gene segment rearranged with a human Jk5 gene segment or a human Jk1 gene segment.

The present invention provides anti-CD3/anti-MUC16 bispecific molecules, wherein the first antigen binding domain that specifically binds CD3 comprises any of the HCVR amino acid sequences, any of the LCVR amino acid sequences, any of the HCVR/LCVR amino acid sequence pairs, any of the heavy chain CDR1-CDR2-CDR3 sequences or any of the light chain amino acid sequences CDR1-CDR2-CDR3 as set forth in US Publication No. 2014/0088295 published March 27, 2014, and PCT/US2016/044732 filed July 29, 2016.

In addition, the present invention provides anti-CD3/anti-MUC16 bispecific molecules in which the first antigen binding domain that specifically binds CD3 comprises any of the HCVR amino acid sequences as set forth in Table. 16, 18 and 22 in this document. The first antigen binding domain, which specifically binds CD3, may also comprise any of the LCVR amino acid sequences as indicated in Table 1. 1, 16, 19 and 23 in this document. In certain embodiments of the invention, the first antigen binding domain that specifically binds CD3 comprises any of the HCVR/LCVR amino acid sequence pairs as set forth in Table. 16, 18, 19, 22 and 23 in this document. The present invention also provides bispecific anti-CD3/anti-MUC16 molecules in which the first antigen binding domain that specifically binds CD3 comprises any of the heavy chain amino acid sequences CDR1-CDR2-CDR3 as set forth in Table 1. 16, 18, and 22 herein, and/or any of the light chain amino acid sequences CDR1-CDR2-CDR3 as set forth in Table. 1, 16, 19 and 23 in this document.

In accordance with some embodiments of the invention, the present invention provides anti-CD3/anti-MUC16 bispecific molecules, wherein the first antigen binding domain that specifically binds CD3 comprises a heavy chain variable region (HCVR) having the amino acid sequence as set forth in Table. 16, 18, and 22 herein, or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also provides anti-CD3/anti-MUC16 bispecific molecules in which the first antigen binding domain that specifically binds CD3 comprises a light chain variable region (LCVR) having the amino acid sequence as set forth in Table 1. 1, 6, 19, and 23 herein, or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

- 9 045730

The present invention also provides aHTu-CD3/aHTu-MUC16 bispecific molecules, in which the first antigen binding domain that specifically binds CD3 contains a pair of amino acid sequences HCVR and LCVR (HCVR/LCVR), as indicated in table. 16, 18, 19, 22 and 23 in this document.

The present invention also provides anti-CD3/anti-MUC16 bispecific molecules in which the first antigen binding domain that specifically binds CD3 comprises a heavy chain CDR3 domain (HCDR3) having the amino acid sequence as set forth in Table 1. 16, 18 and 22 herein, or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a light chain CDR3 domain (LCDR3) having an amino acid sequence as shown in table. 1, 16, 19 and 23 herein, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

In some embodiments, the first antigen binding domain that specifically binds CD3 comprises the amino acid sequence pair HCDR3/LCDR3, as set forth in Table. 16, 18, 19, 22 and 23 in this document.

The present invention also provides anti-CD3/anti-MUC16 bispecific antigen binding molecules, wherein the first antigen binding domain that specifically binds CD3 comprises a heavy chain CDR1 domain (HCDR1) having the amino acid sequence as set forth in Table. 16, 18, and 22 herein, or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; heavy chain CDR2 domain (HCDR2) having an amino acid as indicated in table. 16, 18 and 22, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; heavy chain CDR3 domain (HCDR3) having an amino acid as indicated in table. 16, 18 and 22, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; light chain CDR1 (LCDR1) domain having the amino acid sequence as indicated in table. 1, 16, 19 and 23 herein, or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; light chain CDR2 domain (LCDR2) having the amino acid sequence as indicated in table. 1, 16, 19 and 23 herein, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a light chain CDR3 domain (LCDR3) having an amino acid sequence as shown in table. 1, 16, 19 and 23 herein, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

Some non-limiting, illustrative anti-CD3/anti-MUC16 bispecific antigen binding molecules of the present invention include a first antigen binding domain that specifically binds CD3 containing HCDR1-HCDR2-HCDR3-LCDR1-LCDR2LCDR3 domains, respectively having the amino acid sequences as indicated in table. 16, 18, 19, 22 and 23 in this document.

The present invention also provides a bispecific antigen binding molecule wherein the first antigen binding domain that specifically binds human CD3 comprises the heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) from the heavy chain variable region (HCVR) containing the amino acid sequence as set forth in table 16, table. 18, or table. 22, and the light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) from the light chain variable region (LCVR) containing the amino acid sequence as shown in table. 1, table. 16, table. 19 or table. 23.

In another aspect, the present invention provides a bispecific antigen binding molecule wherein the first antigen binding domain that specifically binds to human CD3 comprises heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) from a heavy chain variable region (HCVR) selected from the group consisting of of SEQ ID NO: 1730, 1762, 1778, 1786 and 1866, and the light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) from the light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 26.

The invention further provides a bispecific antigen binding molecule in which the first antigen binding domain that specifically binds to human CD3 contains three heavy chain complementarity determining regions (A1-HCDR1, A1-HCDR2 and A1-HCDR3) and three light chain complementarity determining regions (A1-HCDR1, A1-HCDR2 and A1-HCDR3). A1-LCDR1, A1-LCDR2 and A1-LCDR3), wherein A1-HCDR1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1732, 1764, 1780, 1788 and 1868; A1-HCDR2 contains the amino acid sequence

- 10 045730 selected from the group consisting of SEQ ID NO: 1734, 1766, 1782, 1790 and 1870; A1-HCDR3 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 1736, 1768, 1784, 1792 and 1872; A1-LCDR1 contains the amino acid sequence SEQ ID NO: 28; A1-LCDR2 contains the amino acid sequence SEQ ID NO: 30; and A1-LCDR3 contains the amino acid sequence of SEQ ID NO: 32.

In yet another aspect, the present invention provides a bispecific antigen binding molecule, wherein the first antigen binding domain that specifically binds human CD3 comprises a heavy and light chain CDR of the HCVR/LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NO: 1730 /26, 1762/26, 1778/26, 1786/26 and 1866/26.

In another aspect, the present invention provides a bispecific antigen binding molecule wherein the first antigen binding domain that specifically binds to human CD3 comprises three heavy chain complementarity determining regions (A1-HCDR1, A1-HCDR2 and A1-HCDR3) and three light chain complementarity determining regions (A1-LCDR1, A1-LCDR2 and A1LCDR3), and wherein the second antigen binding domain, which specifically binds human MUC16, contains three heavy chain complementarity determining regions (A2-HCDR1, A2-HCDR2 and A2-HCDR3) and three light chain complementarity determining regions (A2-LCDR1, A2-LCDR2 and A2LCDR3); wherein A1-HCDR1 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 1732, 1764, 1780, 1788 and 1868; A1-HCDR2 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 1734, 1766, 1782, 1790 and 1870; A1-HCDR3 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 1736, 1768, 1784, 1792 and 1872; A1-LCDR1 contains the amino acid sequence SEQ ID NO: 28; A1LCDR2 contains the amino acid sequence SEQ ID NO: 30; and A1-LCDR3 contains the amino acid sequence of SEQ ID NO: 32; and wherein A2-HCDR1 comprises the amino acid sequence of SEQ ID NO: 20; A2-HCDR2 contains the amino acid sequence SEQ ID NO: 22; A2-HCDR3 contains the amino acid sequence SEQ ID NO: 24; A2-LCDR1 contains the amino acid sequence SEQ ID NO: 28; A2-LCDR2 contains the amino acid sequence SEQ ID NO: 30; and A2-LCDR3 contains the amino acid sequence of SEQ ID NO: 32.

Some non-limiting, illustrative anti-CD3/anti-MUC16 bispecific antigen binding molecules of the present invention include a first antigen binding domain that specifically binds to a CD3 heavy chain containing variable domain framework regions having an amino acid sequence selected from FR1 (SEQ ID NO: 1903), FR2 (SEQ ID NO: 1904), FR3 (SEQ ID NO: 1905) and FR4 (SEQ ID NO: 1906).

In several embodiments, exemplary anti-CD3/anti-MUC16 bispecific antigen binding molecules of the present invention include a bispecific antigen binding molecule wherein the first antigen binding domain that specifically binds human CD3 comprises an HCVR comprising HCDR1-HCDR2-HCDR3, having the amino acid sequences SEQ ID NO: 1907-1908-1909.

The present invention also provides anti-CD3/anti-MUC16 bispecific molecules, wherein the second antigen binding domain that specifically binds MUC16 comprises a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 2 , 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 202, 218, 234, 250, 258, 274, 282, 298, 314, 330, 346, 362 and 378 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also provides anti-CD3/anti-MUC16 bispecific molecules, wherein the second antigen binding domain that specifically binds MUC16 comprises a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 10 ; 26; 42; 58, 74; 90; 106; 122; 138; 154; 170; 186; 210; 226, 242; 266; 290; 306; 322; 338; 354; 370; 386; 1936; and 394, or a substantially similar sequence thereof, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also provides anti-CD3/anti-MUC16 bispecific molecules, wherein the second antigen binding domain that specifically binds MUC16 comprises a pair of amino acid sequences HCVR and LCVR (HCVR/LCVR) selected from the group consisting of SEQ ID NO: 18 /26.

The present invention also provides anti-CD3/anti-MUC16 bispecific molecules, wherein the second antigen binding domain that specifically binds to MUC16 comprises a heavy chain CDR3 domain (HCDR3) having an amino acid sequence selected from the group consisting of SEQ ID NO: 8 , 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 208, 224, 240, 256, 264, 280, 288, 304, 320, 336, 352, 368 and 384 , or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98%, or at least 99% identical

- 11 045730 consistency of the sequence; and a light chain CDR3 (LCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 216, 232 , 248, 272, 296, 312, 328, 344, 360, 376, 392, 400 and 1942, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

In some embodiments, the second antigen binding domain that specifically binds MUC16 comprises the amino acid sequence pair HCDR3/LCDR3 selected from the group consisting of SEQ ID NO: 24/32.

The present invention also provides anti-CD3/anti-MUC16 bispecific antigen binding molecules, wherein the second antigen binding domain that specifically binds MUC16 comprises a heavy chain CDR1 domain (HCDR1) having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 204, 220, 236, 252, 260, 276, 284, 300, 316, 332, 348, 364 and 380, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; a heavy chain CDR2 (HCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 206, 222, 238, 254, 262, 278, 286, 302, 318, 334, 350, 366 and 382, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 208, 224, 240, 256, 264, 280, 304, 320, 336, 352, 368 and 384, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least at least 99% sequence identity; light chain CDR1 (LCDR1) domain having an amino acid sequence selected from the group consisting of SeQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 396, 212, 228, 244, 396, 268, 396, 292, 308, 324, 340, 356, 372, 1938 and 388, or a substantially similar sequence having at least 90%, at least 95%, at least 98 % or at least 99% sequence identity; and a light chain CDR2 domain (LCDR2) having an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 398, 214 , 230, 246, 398, 270, 398, 294, 310, 326, 342, 358, 374, 1940 and 390, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a light chain CDR3 (LCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 400, 216 , 232, 248, 400, 272, 400, 296, 312, 328, 344, 360, 376, 1942 and 392, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

Some non-limiting, illustrative anti-CD3/anti-MUC16 bispecific antigen binding molecules of the present invention include a second antigen binding domain that specifically binds to MUC16 containing HCDR1-HCDR2-HCDR3-LCDR1-LCDR2LCDR3 domains, respectively, having amino acid sequences selected from the group consisting of: SEQ ID NO: 20-22-24-28-30-32.

In a related embodiment, the present invention includes anti-CD3/anti-MUC16 bispecific antigen-binding molecules, wherein the second antigen-binding domain that specifically binds to MUC16 comprises heavy and light chain CDR domains contained in heavy and light chain variable region (HCVR) sequences. LCVR) selected from the group consisting of SEQ ID NO: 18/26.

In one embodiment, the present invention provides an anti-CD3/anti-MUC16 bispecific antibody comprising an anti-MUC16 binding arm that contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 1959 and a light chain containing the amino acid sequence of SEQ ID NO: 1959. 1960, and an anti-CD3 binding arm that contains a heavy chain containing the amino acid sequence of SEQ ID NO: 1961, and a light chain containing the amino acid sequence of SEQ ID NO: 1960. In another embodiment, the present invention provides anti-CD3/anti- MUC16 is a bispecific antibody comprising an anti-MUC16 binding arm that contains a heavy chain containing the amino acid sequence of SEQ ID NO: 1959 and a light chain containing the amino acid sequence of SEQ ID NO: 1960, and an anti-CD3 binding arm that contains the heavy a chain containing the amino acid sequence of SEQ ID NO: 1962, and a light chain containing the amino acid sequence of SEQ ID NO: 1960.

In another aspect, the present invention provides a bispecific antigen binding molecule comprising a first antigen binding domain that binds to human CD3 and a second antigen binding domain that binds to human MUC16, wherein the second antigen binding

- 12 045730 This domain is derived from an antibody or antigen binding fragment of any of the anti-MiC16 antibodies of this invention. In a further aspect, the present invention provides a bispecific antigen binding molecule comprising a first antigen binding domain that specifically binds to human CD3 and a second antigen binding domain that specifically binds to human MUC16.

The invention further provides a bispecific antigen-binding molecule that binds to a human cell expressing human CD3 and a cynomolgus cell expressing cynomolgus CD3. In another aspect, the bispecific antigen binding molecule binds to human cells expressing human MUC16.

In another aspect, the present invention provides a bispecific antigen-binding molecule that inhibits tumor growth in immunodeficient mice bearing human ovarian cancer xenografts. The present invention further provides a bispecific antigen-binding molecule that inhibits tumor growth of established tumors in immunocompromised mice bearing human ovarian cancer xenografts.

In another aspect, the present invention provides a bispecific antigen binding molecule comprising: i) a first antigen binding domain that specifically binds to an effector cell with an EC ₅₀ value of greater than about 4 nM and, and ii) a second antigen binding domain that specifically binds to a target ovarian tumor cell human with an _EC50 value of less than 3 nM, such an _EC50 binding value being measured in an in vitro FACS binding assay.

In one embodiment of the invention, the bispecific antigen binding molecule may include a second antigen binding domain that specifically binds to a target ovarian tumor cell with an _EC50 value of less than about 2 nM. In some cases, the first antigen binding domain specifically binds to each of human CD3 and cynomolgus CD3 with an EC ₅₀ value of greater than about 40 nM, greater than about 100 nM, greater than about 200 nM, greater than about 300 nM, greater than about 400 nM , more than about 500 nM or more than about 1 μM. In some cases, the first antigen binding domain specifically binds to each of human CD3 and cynomolgus CD3 with weak or no measurable binding or binding affinity.

In some embodiments, the antigen binding molecule induces T cell killing of tumor cells with an EC ₅₀ value of less than about 31 pM, as measured in an in vitro T cell killing of tumor cells assay, for example, where the tumor cells are OVCAR3 cells.

In some applications, the first antigen binding domain binds to human CD3 with a KD value of greater than about 11 nM, as measured in an in vitro surface plasmon resonance binding assay. In some cases, the first antigen binding domain binds to each of human CD3 and cynomolgus CD3 with a KD value of greater than about 15 nM, greater than about 30 nM, greater than about 60 nM, greater than about 120 nM, greater than about 300 nM, or more than about 500 nM, as measured in an in vitro surface plasmon resonance binding assay.

In some embodiments, the anti-CD3 antibodies of the present invention, antigen binding fragments, and bispecific antibodies thereof were made by replacing parent amino acid residues in a stepwise manner based on differences between the germline sequence and the parent antibody sequence.

In some embodiments, the present invention provides a bispecific antigen binding molecule in which a second antigen binding domain competes for binding to human MUC16 with a reference antigen binding protein containing three heavy chain complementarity determining regions (A2-HCDR1, A2-HCDR2 and A2-HCDR3) and three light chain region complementarity (A2-LCDR1, A2-LCDR2 and A2-LCDR3), where A2-HCDR1 contains the amino acid sequence SEQ ID NO: 2 0; A2-HCDR2 contains the amino acid sequence SEQ ID NO: 22; A2-HCDR3 contains the amino acid sequence SEQ ID NO: 24; A2-LCDR1 contains the amino acid sequence SEQ ID NO: 28; A2-LCDR2 contains the amino acid sequence SEQ ID NO: 30; and A2-LCDR3 comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the present invention provides a bispecific antigen binding molecule wherein a second antigen binding domain competes for binding to human MUC16 with a reference antigen binding protein containing a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 18, and a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the present invention provides a bispecific antigen binding molecule wherein the first antigen binding domain competes for binding to human CD3 with a reference antigen binding protein containing three heavy chain complementarity determining regions (A1-HCDR1, A1-HCDR2 and A1-HCDR3) and three light chain region complementarity (A1-LCDR1, A1-LCDR2 and A1-LCDR3), A1-HCDR1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1732, 1764, 1780, 1788 and 1868;

- 13 045730

A1-HCDR2 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 1734, 1766, 1782, 1790 and 1870; A1-HCDR3 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 1736, 1768, 1784, 1792 and 1872; A1-LCDR1 contains the amino acid sequence SEQ ID NO: 28; A1-LCDR2 contains the amino acid sequence SEQ ID NO: 30; and A1-LCDR3 comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the present invention provides a bispecific antigen binding molecule wherein the first antigen binding domain competes for binding to human CD3 with a reference antigen binding protein containing a heavy chain variable region (HCVR) containing an amino acid sequence selected from the group consisting of SEQ ID NO: 1730, 1762, 1778, 1786 and 1866, and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the present invention provides a bispecific antigen binding molecule wherein the first antigen binding domain competes for binding to human CD3 with a reference antigen binding protein containing a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1730, 1762, 1778, 1786 and 1866, and a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 26; and wherein the second antigen binding domain competes for binding to human MUC16 with a reference antigen binding protein comprising a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18 : 26.

In one aspect, the present invention provides a pharmaceutical composition comprising an anti-MUC16 antigen binding molecule or an anti-MUC16/anti-CD3 bispecific antigen binding molecule and a pharmaceutically acceptable carrier or diluent. The present invention further provides a method of treating cancer in a subject, comprising administering to the subject a pharmaceutical composition comprising an anti-MUC16 antigen binding molecule or an anti-MUC16/antiCD3 bispecific antigen binding molecule and a pharmaceutically acceptable carrier or diluent. In some embodiments, the cancer is selected from the group consisting of ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, mass-forming intrahepatic cholangiocarcinoma, cervical adenocarcinoma, and gastric adenocarcinoma. In some cases, the cancer is ovarian cancer.

In another aspect, the present invention relates to nucleic acid molecules encoding any of the HCVR, LCVR or CDR sequences of the anti-CD3/anti-MUC16 bispecific antigen-binding molecules described herein, including nucleic acid molecules containing polynucleotide sequences as set forth in table. 2, 17, 20, 21, 23 and 25 herein, as well as nucleic acid molecules comprising two or more polynucleotide sequences, as indicated in table. 2, 17, 20, 21, 23 and 2 5 in any functional combination or arrangement. Recombinant expression vectors carrying the nucleic acids of the present invention and the host cells into which such vectors have been introduced are also included in the invention, as are methods for producing antibodies by culturing the host cells under conditions allowing the production of antibodies and reconstituting the antibodies produced.

This invention includes anti-CD3/anti-MUC16 bispecific antigen binding molecules, wherein any of the above antigen binding domains that specifically bind CD3 are combined, linked or otherwise linked to any of the above antigen binding domains that specifically bind MUC16 to form a bispecific antigen-binding molecule that binds to CD3 and MUC16.

The present invention includes anti-CD3/anti-MUC16 bispecific antigen binding molecules having a modified glycosylation pattern. In some applications, modification to remove unwanted glycosylation sites may be useful, or the antibody lacking the fucose moiety present in the oligosaccharide chain, for example, to enhance the function of antibody-dependent cellular cytotoxicity (ADCC) (see Shield et al. (2002) JBC 277: 26733). In other applications, modification of galactosylation can be made to alter complement-dependent cytotoxicity (CDC).

In another aspect, the present invention provides a pharmaceutical composition comprising an anti-CD3/anti-MUC16 bispecific antigen binding molecule as described herein and a pharmaceutically acceptable carrier. In a related aspect, the present invention provides a composition that is a combination of an anti-CD3/anti-MUC16 bispecific antigen binding molecule and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-CD3/anti-MUC16 bispecific antigen-binding molecule. Exemplary agents that may advantageously be combined with anti-CD3/anti-MUC16 bispecific antigen binding

- 14 045730 by this molecule are discussed in detail elsewhere in this document.

In yet another aspect, the present invention relates to therapeutic methods for targeting/killing tumor cells expressing MUC16 using an anti-CD3/anti-MUC16 bispecific antigen binding molecule of the present invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising anti-CD3 /anti-MUC16 bispecific antigen binding molecule of the present invention, to a subject in need thereof.

The present invention also includes the use of the anti-CD3/anti-MUC16 bispecific antigen binding molecule of the present invention in the manufacture of a medicament for the treatment of a disease or disorder associated with or caused by MUC16-expressing cells.

In another aspect, the present invention provides a method for detecting MUC16 in a biological sample, comprising: obtaining a biological sample from a subject, and detecting whether MUC16 is present in the biological sample by contacting the biological sample with an anti-MUC16 antibody or antigen-binding fragment thereof and detecting binding between MUC16 and anti-MUC16 antibody or antigen binding fragment. In some cases, the biological sample is a tissue or fluid sample selected from plasma, serum, ascites, ovary, uterus, cervix, liver, bladder, pancreas, stomach, small or large intestine, gallbladder, breast, lung, kidney, saliva, and lacrimal glands, or any epithelioid malignancy thereof. In some embodiments, the antibody or antigen binding fragment binds human MUC16 at one or more of the five membrane-proximal SEA domains of human MUC16 corresponding to residues 1379114451 of SEQ ID NO: 1899. In some embodiments, the antibody or antigen binding fragment thereof binds human MUC16 at residues 13810-14451 SEQ ID NO: 1899. In some embodiments, the antibody or antigen binding fragment thereof binds to any of several SEA1, SeA2, SEA3, SEA4, SEA5, SEA6, SEA7, SEA8, SEA9, SEA10, SEA11, SEA12 , SEA13, SEA14, SEA15 or SEA16 MUC16 person.

In another aspect, the present invention provides a method for detecting MUC16 in a patient, comprising: obtaining a tissue sample from the patient; and detecting the presence of MUC16 in a tissue sample by contacting the tissue sample with an anti-MUC16 antibody and detecting binding between MUC16 and the anti-MUC16 antibody. In some cases, the method further includes diagnosing a cancer patient when the presence of MUC16 is detected in the tissue sample. In one embodiment of the invention, the tissue sample is ovarian tissue. In some cases, the anti-MUC16 antibody is specific for an epitope at residues 12783-13467 of SEQ ID NO: 1899. In one embodiment, the anti-MUC16 antibody comprises a CDR of the HCVR/LCVR pair containing the amino acid sequences of SEQ ID NO: 202/210. In some cases, the anti-MiC16 antibody is specific for an epitope at residues 1381014451 of SEQ ID NO: 1899. In one embodiment, the anti-MiC16 antibody comprises a CDR of the HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO: 250 /1936, 258/266, 314/322 and 1944/1952.

In another aspect, the present invention provides a method for detecting MUC16 in a patient, comprising: obtaining a plasma sample from the patient; and detecting the presence of MUC16 in a plasma sample by contacting the plasma sample with an anti-MiC16 antibody comprising a CDR of an HCVR/LCVR pair comprising the amino acid sequences of SEQ ID NO: 202/210, and detecting binding between MUC16 and the anti-MiC16 antibody. In some cases, the method further includes diagnosing a cancer patient when the presence of MUC16 is detected in the plasma sample. In some embodiments, the method further includes administering an effective amount of a bispecific anti-CD3xMUC16 antibody to a diagnosed patient. In some embodiments, the method further comprises administering an effective amount of an ADC comprising an anti-MiC16 antibody or an antigen-binding fragment thereof and a cytotoxic agent to the diagnosed patient.

In another aspect, the present invention provides an isolated antibody or antigen binding fragment thereof that binds to MUC16, wherein the antibody or antigen binding fragment thereof comprises a CDR of the amino acid sequence pair HCVR/LCVR selected from the group consisting of SEQ ID NO: 202/210, 250/ 1936, 258/266, 314/322, 82/858, 98/170 and 1944/1952. In some embodiments, the antibody or antigen binding fragment thereof comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, selected from the group consisting of SEQ ID NO: 204-206-208-212-214-216; 252-254-256-1938-1940-1942; 260-262-264-268-270-272; 316-318-320-324-326-328; 84-86-88-1892-1894-1896; 100-102-104-172-174-176; and 1946-1948-1950-19541956-1958. In some embodiments, the antibody or antigen binding fragment thereof comprises a paired HCVR/LCVR amino acid sequence selected from the group consisting of 202/210, 250/1936, 258/266, 314/322, 82/858, 98/170, and 1944/1952.

Other embodiments of the present invention will become apparent from a review of the following detailed description.

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Brief description of graphic materials

Fig. 1-3 illustrate the pharmacokinetic profiles of anti-MiC16 x CD3 bispecific antibodies in wild-type mice (Fig. 1), humanized CD3 mice (Fig. 2) or humanized MUC16 x CD3 mice (Fig. 3).

In fig. Figure 4 shows the results of study 1 of the OVCAR-3 model (average radiation [f/s/cm ² /sr] on day 6). All groups had similar tumor burden as assessed by BLI before dosing. Data shown are tumor burden assessed by BLI at day 6 after tumor implantation. Statistical significance was determined using unpaired nonparametric Mann-Whitney t tests. There was no significant difference in tumor burden between groups.

In fig. Figure 5 shows the results of study 1 of the OVCAR-3 model (average radiation [f/s/cm ² /sr] on day 20). BSMUC16/CD3-001 significantly reduced tumor burden at 0.1 and 0.5 mg/kg. NSG mice implanted with human T cells were implanted with human OVCAR3/Luc cells. Treatment began 6 days after tumor implantation. Mice were treated on days 6, 10, 13, 16, and 21 with 0.01, 0.1, or 0.5 mg/kg BSMUC16/CD3-001 administered intraperitoneally, or treated with a CD3 binding control or no binding control (0. 5 mg/kg intraperitoneally). Data shown are tumor burden assessed by BLI at day 20 after tumor implantation. Statistical significance was determined using unpaired nonparametric Mann-Whitney t tests. Treatment with BSMUC16/CD3-001 was compared with a no-binding control (** p < 0.01 for 0.5 mg/kg, # p < 0.05 for 0.1 mg/kg BSMUC16/CD3-001).

In fig. Figure 6 shows the results of Study 1 of the OVCAR-3 model (fold change in BLI-proven tumors between D6 and D20). BSMUC16/CD3-001 significantly reduced the fold change in tumor burden at 0.01, 0.1 and 0.5 mg/kg. NSG mice implanted with human T cells were implanted with human OVCAR-3/Luc cells. Mice were treated on days 6, 10, 13, 16, and 21 with 0.01, 0.1, or 0.5 mg/kg BSMUC16/CD3-001 administered intraperitoneally, or treated with a CD3 binding control or no binding control (0. 5 mg/kg intraperitoneally). Data shown represent the fold change in tumor burden from the first measurement (taken before treatment) to day 20 at the end of the study. Statistical significance was determined using unpaired nonparametric Mann-Whitney t tests. Treatment with BSMUC16/CD3-001 was compared with no-binding control (** p < 0.01 for 0.5 mg/kg, # p < 0.05 for 0.1 mg/kg, $ p < 0.05 for 0.01 mg/kg BSMUC16/CD3-001).

In fig. Figure 7 shows the results of Study 2 of the OVCAR-3 model (average irradiance [f/s/cm ² /sr] on day 4). All groups had similar tumor burden as assessed by BLI before dosing. Data shown are tumor burden assessed by BLI at day 4 after tumor implantation. Statistical significance was determined using unpaired nonparametric Mann-Whitney t tests. There was no significant difference in tumor burden at day 4 between groups.

In fig. Figure 8 shows the results of Study 2 of the OVCAR-3 model (average radiation [f/s/cm ² /sr] on day 25). BSMUC16/CD3-005 significantly reduced tumor burden at 0.5, 1 and 5 mg/kg. NSG mice implanted with human T cells were implanted with human OVCAR3/Luc cells. Treatment began 5 days after tumor implantation. Mice were treated on days 5, 8, 12, 15, 19, and 22 with 0.1, 0.5, 1, or 5 mg/kg REGN4019 administered i.v., or administered a CD3 binding control or no binding control (5 mg/kg). kg i.v.). Data shown are tumor burden assessed by BLI at day 25 after tumor implantation. Statistical significance was determined using unpaired nonparametric Mann-Whitney t tests. Treatment with BSMUC16/CD3-005 was compared with control without binding (** p < 0.01 for 5 mg/kg, ## p < 0.01 for 1 mg/kg, $$ p < 0.01 for 0. 5 mg/kg BSMUC16/CD3-005).

In fig. Figure 9 shows the results of Study 2 of the OVCAR-3 model (fold change in BLI-proven tumors between D4 and D25). BSMUC16/CD3-005 significantly reduced tumor growth at 0.5, 1 and 5 mg/kg. NSG mice implanted with human T cells were implanted with human OVCAR-3/Luc cells. Mice were treated on days 5, 8, 12, 15, 19, and 22 with 0.1, 0.5, 1, or 5 mg/kg REGN4019 administered i.v., or treated with CD3-binding control or no-binding control (5 mg/kg i.v.). Data shown are the fold change in tumor burden from the first measurement (taken the day before treatment) to day 25 at the end of the study. Statistical significance was determined using unpaired nonparametric Mann-Whitney t tests. Treatment with BSMUC16/CD3-005 was compared with control without binding (** p < 0.01 for 5 mg/kg, ## p < 0.01 for 1 mg/kg, $$ p < 0.01 at 0. 5 mg/kg REGN4019).

In fig. Figure 10 shows the results of the ID8-VEGF/huMUC16 model. Tumor size at 4 to 7 days after implantation BSMUC16/CD3-001 significantly reduces tumor growth in a syngeneic model when treatment begins either on the day of implantation or 10 days after tumor implantation Mice expressing human CD3 instead of murine CD3 and a chimeric MUC16 molecule were implanted

- 16 045730 mouse ovarian tumor line expressing part of human MUC16. Mice were treated with BSMUC16/CD3-001 (100 μg i.p.) on the day of implantation or 10 days postimplantation or treated with CD3-binding control (100 μg i.p.) on the day of implantation. Mice were treated on days 0, 4, 7, 10, 13, 17, 20, or 2 4 for the rescue groups and on days 10, 13, 17, 20, and 24 for the group where dosing began on D10. Data shown are tumor volumes at day 47 postimplantation. Statistical significance was determined using unpaired nonparametric Mann-Whitney t tests. Treatment with BSMUC16/CD3-001 was compared with a CD3 binding control (** p < 0.01 from D0, * p < 0.05 from D10).

Fig. 11A-11C illustrate results of flow cytometry (or FACS) analysis of binding of selected bispecific antibodies to PEO-1, OVCAR3-Luc, Jurkat cells, and cynomolgus T cells. Titration assays were performed by testing a series of serial dilutions of each antibody: either the MUC16xCD3 bispecific antibodies BSMUC16/CD3-001, BSMUC16/CD3-002, or BSMUC16/CD3-003, or the first or second isotype control antibody (not cross-reactive to CD3 or MUC16 ).

Fig. 12A and 12B depict examples of killing of PEO-1 (FIG. 12A) or OVCAR3-Luc (FIG. 12B) cells in a 48 hour cytotoxicity assay by anti-MUC16 x anti-CD3 treatment in the presence of human PBMCs.

Detailed description of the invention

Before describing the present invention, it should be understood that the present invention is not limited to the specific methods described and the experimental conditions described, since such methods and conditions may vary. It should also be understood that the terminology used herein is intended solely to describe specific embodiments of this invention and is not limiting, since the scope of this invention is limited only by the accompanying claims.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention relates. As used herein, the term about, when used in relation to a specific numerical value given, means that the value may differ from the given value by no more than 1%. For example, as used herein, the expression about 100 includes 99 and 101 and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods and materials are now described. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

Definitions

The expression CD3, as used herein, refers to an antigen that is expressed on T cells as part of the multimolecular T-cell receptor (TCR), and which consists of a homodimer or heterodimer formed from the association of two of the four chains of the receptor: CD3epsilon, CD3- delta, CD3-zeta and CD3-gamma. Human CD3 epsilon contains the amino acid sequence shown in SEQ ID NO: 18 97; Human CD3 delta contains the amino acid sequence set forth in SEQ ID NO: 1898. All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human version of the corresponding protein, polypeptide, or protein fragment unless specifically stated to refer to to a non-human species. Thus, the expression CD3 means human CD3 unless specified to be of a non-human species, such as mouse CD3, monkey CD3, etc.

As used herein, the term antibody that binds to CD3 or anti-CD3 antibody includes antibodies and antigen binding fragments that specifically recognize one CD3 subunit (e.g., epsilon, delta, gamma, or zeta), as well as antibodies and antigen binding fragments thereof that that specifically recognize a dimeric complex of two CD3 subunits (eg, gamma/epsilon, delta/epsilon, and zeta/zeta CD3 dimers). The antibodies and antigen binding fragments of this invention can bind soluble CD3 and/or cell surface expressed CD3. Soluble CD3 includes natural CD3 proteins as well as variants of recombinant CD3 proteins, such as, for example, monomeric and dimeric CD3 constructs that do not have a transmembrane domain or are otherwise not associated with the cell membrane.

As used herein, the expression CD3 expressed on the cell surface means one or more CD3 protein(s) that is/are expressed on the cell surface in vitro or in vivo such that at least a portion of the CD3 protein is exposed on the extracellular side of the cell. membrane and is accessible to the antigen-binding portion of the antibody. CD3 expressed on the cell surface includes CD3 proteins contained in the context of a functional T cell receptor in the cell membrane. Cell surface expressed CD3 includes CD3 protein expressed as part of a cell surface homodimer or heterodimer (eg, gamma/epsilon, delta/epsilon, and zeta/zeta CD3 dimers). CD3 expression, ex

- 17 045730 cell surface pressable also includes a CD3 chain (eg CD3 epsilon, CD3 delta or CD3 gamma) that is expressed by itself, without other types of CD3 chain, on the cell surface. CD3 expressed on the cell surface may include or consist of CD3 protein expressed on the surface of a cell that normally expresses CD3 protein. Alternatively, CD3 expressed on the cell surface may include or consist of a CD3 protein expressed on the surface of a cell that does not normally express human CD3 on its surface but has been artificially engineered to express CD3 on its surface.

The expression MUC16 as used herein refers to mucin 16. MUC16 is a single transmembrane domain highly glycosylated integral membrane glycoprotein that is highly expressed in ovarian cancer. The amino acid sequence of human MUC16 is shown in SEQ ID NO: 1899.

As used herein, the term antibody that binds MUC16 or anti-MUC16 antibody includes antibodies and antigen-binding fragments thereof that specifically recognize MUC16.

The term antigen-binding molecule includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

The term antibody as used herein means any antigen-binding molecule or molecular complex containing at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (eg, MUC16 or CD3). The term antibody includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds, as well as their multimers (for example, IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2 and _CH3 . Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region ( _CL ). The light chain constant region contains one domain (CL1). The V _H and V _L regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FR). Each V _H and V _L consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the invention, the FR of an anti-MUC16 antibody or an anti-CD3 antibody (or an antigen-binding portion thereof) may be identical to human germline sequences, or may be naturally or artificially modified. A consensus amino acid sequence can be determined based on parallel analysis of two or more CDRs.

The term antibody as used herein also includes antigen-binding fragments of complete antibody molecules. The terms antigen binding portion of an antibody, antigen binding fragment of an antibody, and the like as used herein include any naturally occurring, enzymatically produced, synthetic or genetically modified polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding antibody fragments can be prepared, for example, from full-length antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and optionally constant domains of the antibody. Such DNA is known and/or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA may be sequenced and processed chemically or using molecular biology techniques, for example, to organize one or more variable and/or constant domains into a suitable configuration or to introduce codons, create cysteine residues, modify, add or delete amino acids, and the like.

Non-limiting examples of antigen binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic a hypervariable region of an antibody (eg, a dedicated complementarity determining region (CDR) such as a CDR3 peptide) or a conformationally restricted peptide FR3-CDR3-FR4. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tri-bodies, tetrabodies, mini-bodies, nanobodies (e.g., monovalent nanobodies, divalent nanobodies, and the like), immunopharmaceuticals small modular protein (SMIP)-based agents and shark IgNAR variable domains are also encompassed by the expression antigen binding fragment as used herein.

The antigen-binding fragment of an antibody typically contains at least one variable

- 18 045730 domain. The variable domain can be of any size or amino acid composition and typically comprises at least one CDR that is adjacent or in frame to one or more framework sequences. In antigen binding fragments having a _VH domain associated with a _VL domain, the VH and _VL domains may be arranged relative to each other in any suitable order. For example, the variable region may be dimeric and contain dimers VH-VH, _VH - _VL or _VL - _VL . Alternatively, the antigen binding fragment of the antibody may comprise a monomeric _VH or _VL domain.

In some embodiments of the present invention, the antigen binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen binding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) V _H -C _H 1-C _H 2-C _H 3; (vi) Vh-Ch2-Ch3; (vii) Vh-Cl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Ch3; (xi) Vl-Ch1-Ch2; (xii) Vl-Ch1-Ch2-Ch3; (xiii) V _l -C _h 2-C _h 3; and (xiv) V _L -C _L . In any configuration of variable and constant domains, including any of the exemplary configurations above, the variable and constant domains may either be directly linked to each other or may be linked by a full-length or partial hinge or linker region. The hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, the antigen binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more monomeric V _H or V _L domains (eg, disulfide bond(s)).

As with full antibody molecules, antigen binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen-binding antibody fragment will typically contain at least two different variable domains, with each variable domain being capable of specifically binding to a different antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats described herein, can be adapted for use in the context of an antigen binding fragment of an antibody of the present invention using conventional techniques available in the art.

Antibodies of the present invention may function through complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Complement-dependent cytotoxicity (CDC) refers to the lysis of antigen-expressing cells by the antibody of this invention in the presence of complement. Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcR) (eg, natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on the target cell and thereby leading to lysis of the target cell. CDC and ADCC can be measured using assays that are well known and available in the art. (See, for example, US Patent Nos. 5,500,362 and 5,821,337 and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The antibody constant region is important for the antibody's ability to fix complement and mediate cell-dependent cytotoxicity. Thus, the antibody isotype can be selected based on whether the antibody is desired to mediate cytotoxicity.

In some embodiments of the present invention, the anti-MUC16 monospecific antibodies or anti-MUC16/anti-CD3 bispecific antibodies of the present invention are human antibodies. As used herein, the term human antibody refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo), for example, in the CDR and, in particular, CDR3. However, the term human antibody, as used herein, is not intended to include antibodies in which CDR sequences derived from the germ line of other mammalian species, such as mouse, have been fused to human framework sequences.

The antibodies of this invention may, in some embodiments, be recombinant human antibodies. The term recombinant human antibody, as used herein, is intended to include all human antibodies that are produced, expressed, created or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below) , antibodies isolated from a recombinant, combinatorial library of human antibodies (described further below), antibodies isolated from an animal

- 19 045730 (for example, mouse), which is transgenic for human immunoglobulin genes (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20: 6287-6295), or antibodies produced, expressed, created or isolated by any other method that involves splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments of the invention, such recombinant human antibodies are subject to mutagenesis in vitro (or, when an animal transgenic for human Ig sequences is used, to somatic mutagenesis in vivo) and thus the amino acid sequences of the VH and _VL regions of the recombinant antibodies are sequences which, although derived from and related to human germline VH and _VL sequences, would not naturally exist in the human antibody germline repertoire in vivo.

Human antibodies can exist in two forms, which are associated with hinge heterogeneity. In one form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In the second form, the dimers are not linked through interchain disulfide bonds, and a molecule of about 75-80 kDa is formed, consisting of a covalently linked light and heavy chain (half-antibody). These forms are extremely difficult to separate, even after affinity purification.

The frequency of occurrence of the second form in different isotypes of intact IgG is due to, but is not limited to, structural differences associated with the hinge region of the antibody isotype. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using the human IgG1 hinge. The present invention covers antibodies having one or more mutations in the hinge, CH2 or CH3 region, which may be desirable, for example, in manufacturing, to improve the yield of the desired form of the antibody.

Antibodies of this invention may be isolated antibodies. An isolated antibody, as used herein, means an antibody that has been identified and separated and/or extracted from at least one component of its natural environment. For example, an antibody that has been isolated or removed from at least one component of the body, or from a tissue or cell in which the antibody naturally exists or is produced is an isolated antibody for purposes of this invention. The isolated antibody also includes the antibody in situ in the recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. In certain embodiments of the invention, the isolated antibody may be substantially free of other cellular material and/or chemicals.

The present invention also includes single arm antibodies that bind MUC16. As used herein, the term single-arm antibody means an antigen-binding molecule comprising a heavy chain of one antibody and a light chain of one antibody. The single arm antibodies of the present invention may contain any of the HCVR/LCVR or CDR amino acid sequences as set forth in Table. 1.

Ahtu-MUC16 or anti-MUC16/anti-CD3 antibodies described herein may contain one or more amino acid substitutions, insertions and/or deletions in the framework regions and/or CDR regions of the heavy and light chain variable domains compared to the corresponding sequences germ line from which the antibodies were derived. Such mutations can be readily detected by comparing the amino acid sequences described herein with germline sequences available, for example, from publicly available antibody databases. This invention includes antibodies and antigen binding fragments thereof that are derived from any of the amino acid sequences described herein, wherein one or more amino acids in one or more framework regions and/or CDR regions are mutated to the corresponding germline sequence residue(s), from which the antibody was derived, or with the corresponding residue(s) of another human germline sequence, or with a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to collectively herein as germline mutations). One of skill in the art, starting with the heavy and light chain variable region sequences described herein, can readily produce numerous antibodies and antigen binding fragments that contain one or more individual germline mutations or combinations thereof. In some embodiments, all framework residues and/or CDR residues in the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only the mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2, or

- 20 045730

CDR3. In other embodiments, one or more framework residue(s) and/or CDR residue(s) are mutated with the corresponding residue(s) of another germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was derived). originally received). In addition, antibodies of the present invention may contain any combination of two or more germline mutations within framework regions and/or CDR regions, for example, wherein certain individual residues are mutated to the corresponding residue of a certain germline sequence, while certain other residues that differ from the original germline sequence are retained or mutated with the corresponding residue of another germline sequence. Once produced, antibodies and antigen binding fragments that contain one or more germline mutations can be readily tested for one or more desirable properties, such as improved binding specificity, improved binding (e.g., measured by cell binding titration or FACS binding), or binding affinity. improved or enhanced antagonistic or agonistic biological properties (as appropriate), reduced immunogenicity and the like. Antibodies and antigen binding fragments obtained using this general method are covered by this invention.

The invention also includes anti-MUC16 or anti-MUC16/anti-CD3 antibodies containing variants of any of the HCVR, LCVR and/or CDR amino acid sequences described herein having one or more conservative substitutions. For example, the present invention includes anti-MUC16 or anti-MUC16/anti-CD3 antibodies having the amino acid sequences HCVR, LCVR and/or CDR with, for example, 10 or less, 8 or less, 6 or less, 4 or less, and the like conservative amino acid substitutions in relation to any of the amino acid sequences HCVR, LCVR and/or CDR presented in table. 1 in this document or as described in table. 16, 18, 19, 22 and 23 in this document.

The term epitope refers to an antigenic determinant that interacts with a specific antigen-binding site in a variable region of an antibody molecule known as a paratope. One antigen can have more than one epitope. Thus, different antibodies may bind to different regions on the antigen and may have different biological effects. Epitopes can be conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, the epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

The term substantial identity or substantially identical, when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with corresponding nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is at least an amount of nucleotide sequence identity about 95%, and more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any known sequence identity algorithm, such as FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may, in some cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

As applied to polypeptides, the term substantially similar or substantially similar means that two peptide sequences, when optimally aligned, for example using the GAP or BESTFIT programs using standard gap-opening penalties, have at least 95% sequence identity, even more preferably, sequence at least 98% or 99% identical. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. A conservative amino acid substitution is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution will not significantly change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage sequence identity or degree of similarity can be adjusted upward to correct the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art; see, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, incorporated herein by reference. Examples of groups of amino acids with side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) main side chains: lysine, arginine and histidine; 6) acid boko

- 21 045730 chains: aspartate and glutamate and 7) sulfur-containing side chains - cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix described in Gonnet et al. (1992) Science 256: 1443-1445, incorporated herein by reference. A moderately conservative replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software finds similar sequences using similarity measurements assigned to various substitutions, deletions and other modifications, including conserved amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and its mutein; see for example GCG version 6.1. Polypeptide sequences can also be compared using FASTA with standard or recommended parameters; program in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignment identity and sequence percentage of regions of best overlap between the query sequence and the search sequence (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of this invention to a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN, using default parameters; see, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-402, each of which is incorporated herein by reference.

Germline mutations.

The Ahtu-CD3 antibodies described herein contain one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived.

This invention also includes antibodies and antigen binding fragments thereof that are derived from any of the amino acid sequences described herein, wherein one or more amino acids in one or more framework regions and/or CDR regions are mutated to the corresponding germline sequence residue(s) from which the antibody was derived, or with the corresponding residue(s) of another human germline sequence or with a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as germline mutations) and having weak or undetectable binding with CD3 antigen. Several such typical antibodies that recognize CD3 are described in Table. 16, 18, 19, 22 and 23 in this document.

In addition, antibodies of the present invention may contain any combination of two or more germline mutations within framework regions and/or CDR regions, for example, wherein certain individual residues are mutated to the corresponding residue of a certain germline sequence, while certain other residues that differ from the original germline sequence are retained or mutated with the corresponding residue of another germline sequence. Once produced, antibodies and antigen binding fragments that contain one or more germline mutations can be tested for one or more desired properties, such as improved binding specificity, weak or reduced binding or binding affinity, improved or increased pharmacokinetic properties, reduced immunogenicity, etc. d. Antibodies and antigen binding fragments obtained by this general method in accordance with the teachings of this invention are included in this invention.

The invention also includes anti-CD3 antibodies containing variants of any of the HCVR, LCVR and/or CDR amino acid sequences described herein having one or more conservative substitutions. For example, the present invention includes anti-CD3 antibodies having amino acid sequences HCVR, LCVR and/or CDR with, for example, 10 or less, 8 or less, 6 or less, 4 or less, and the like, conservative amino acid substitutions with respect to any of amino acid sequences HCVR, LCVR and/or CDR presented in table. 16, 18, 19, 22 and 23 in this document. The antibodies and bispecific antigen binding molecules of the present invention contain one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the individual antigen binding domains were derived. maintaining or improving the desired weak or undetectable connection

- 22 045730 calling with CD3 antigen. A conservative amino acid substitution is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution will not significantly alter the functional properties of the protein, that is, the amino acid substitution maintains or improves the desired weak or undetectable binding or binding affinity in the case of anti-CD3 binding molecules. Examples of groups of amino acids with side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) main side chains: lysine, arginine and histidine; 6) acidic side chains: aspartate and glutamate and 7) sulfur-containing side chains - cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate aspartate and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix described in Gonnet et al. (1992) Science 256: 1443-1445. A moderately conservative replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

The invention also includes antigen binding molecules comprising an antigen binding domain with an HCVR and/or CDR amino acid sequence that is substantially identical to any of the HCVR and/or CDR amino acid sequences described herein while maintaining or improving the desired weak affinity for the CD3 antigen. The term substantial identity or substantially identical, when referring to an amino acid sequence, means that two amino acid sequences, when optimally aligned, for example using the GAP or BESTFIT programs using default gap weights, share at least 95% identity sequence, even more preferably at least 98% or 99% sequence identity. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage sequence identity or degree of similarity can be adjusted upward to correct the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art; see, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331.

Sequence similarity for polypeptides, also called sequence identity, is typically measured using sequence analysis software.152 Protein analysis software finds similar sequences using similarity measurements assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and its mutein; see for example GCG version 6.1. Polypeptide sequences can also be compared using FASTA with standard or recommended parameters; program in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignment identity and sequence percentage of regions of best overlap between the query sequence and the search sequence (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of this invention to a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN, using default parameters; see, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-402.

Once obtained, antigen binding domains that contain one or more germline mutations are tested for reduced binding or binding affinity using one or more in vitro assays. Although antibodies that recognize a particular antigen are typically screened for their intended purpose by testing for high (ie, strong) binding or binding affinity to the antigen, the antibodies of the present invention exhibit weak or no binding. Bispecific antigen binding molecules containing one or more antigen binding domains prepared in this general manner are also included in the present invention and have been found to be advantageous as a therapy for avidity driven tumors.

Unexpected benefits, such as improved pharmacokinetic properties and low patient toxicity, may be realized from the methods described herein.

- 23 045730

Binding properties of antibodies.

As used herein, the term binding in the context of binding of an antibody, immunoglobulin, antigen-binding fragment, or Fc-containing protein, or, for example, a predetermined antigen such as a cell surface protein or fragment thereof, generally refers to the interaction or association between at least two objects or molecular structures, such as antibody-antigen interactions.

For example, binding affinity typically corresponds to a K _D value of about 10 ^-7 M or less, for example about 10 ^-8 M or less, for example about 10 ^-9 M or less, when determined, for example, using surface plasmon resonance technology (SPR) in a BIAcore 3000 instrument using an antigen as a ligand and an antibody, Ig, antibody binding fragment, or Fc-containing protein as an analyte (or antiligand). Cellular binding strategies such as fluorescence-activated cell sorting (FACS) are also commonly used, and FACS data correlates well with other methods such as competitive radioligand binding and SPR (Benedict, CA, J. Immunol Methods. 1997, 201(2) : 223-31; Geuijen, CA, et al. J Immunol Methods. 2005, 302 (1-2): 68-77).

Accordingly, the antibody or antigen-binding protein thereof of the present invention binds to a predetermined antigen or cell surface (receptor) molecule having an affinity corresponding to a K _D value that is at least ten times lower than its affinity for binding to a nonspecific antigen (eg , BSA, casein). In accordance with this invention, the affinity of an antibody corresponding to a K _D value that is equal to or less than ten times less than that of a nonspecific antigen can be considered as undetectable binding, however, such an antibody can be paired with a second antigen-binding arm to obtain a bispecific antibody according to this invention.

The term K _D (M) refers to the dissociation of the equilibrium constant of a particular antigen-antibody interaction, or the dissociation of the equilibrium constant of an antibody or antibody binding fragment binding to an antigen. There is an inverse relationship between _KD and binding affinity, so the lower the _KD value, the higher, that is, stronger, the affinity. Thus, the terms higher affinity or stronger affinity refer to a higher ability to form an interaction and therefore a lower value of K _D , and conversely, the terms lower affinity or weaker affinity refer to a lower ability to form an interaction and therefore to a larger value of K _D . In some circumstances, the higher binding affinity (or KD) of a particular molecule (e.g., antibody) to its interactive partner molecule (e.g., antigen X) relative to the binding affinity of a molecule (e.g., antibody) to another interactive partner molecule (e.g., antigen Y) can be expressed as a binding ratio determined by dividing the larger KD value (lower or weaker affinity) by the smaller KD value (higher or stronger affinity), for example, expressed as 5-fold or 10-fold greater affinity binding, depending on the circumstances.

The term K _d (s -1 or 1/s) refers to the dissociation rate constant of a particular antigen-antibody interaction, or the dissociation rate constant of an antibody or antibody-binding moiety. The specified value is also called the kf value.

The term k _a (M-1 x sec-1 or 1/M) refers to the rate constant of association of a particular antibody-antigen interaction, or the rate constant of association of an antibody or antibody-binding moiety.

The term Ka (M-1 or 1/M) refers to the association of the equilibrium constant of a particular antibody-antigen interaction, or the association of the equilibrium constant of an antibody or antibody-binding moiety. The equilibrium constant of the association is obtained by dividing ka by k _d .

The term EC50 or _EC50 refers to the half maximum effective concentration, which includes the concentration of the antibody that induces a response halfway between the baseline and the maximum after a specified exposure time. EC ₅₀ is essentially the concentration of an antibody at which 50% of its maximum effect is observed. In some embodiments, the EC ₅₀ value is equal to the concentration of an antibody of the invention that produces half maximal binding to cells expressing CD3 or tumor associated antigen, as determined, for example, by a FACS binding assay. Thus, reduced or weaker binding is observed as EC ₅₀ or half the maximum effective concentration increases.

In one embodiment of the invention, a decrease in binding can be defined as an increase in the EC ₅₀ concentration of the antibody that allows binding to half the maximum number of target cells.

In another embodiment, the EC ₅₀ value is the concentration of an antibody of the invention that causes half the maximum depletion of target cells by T cell cytotoxic activity. Thus, increased cytotoxicity

- 24 045730 ical activity (eg, T cell-mediated tumor cell death) is observed at a reduced _EC50 value or half the maximum effective concentration.

Bispecific antigen-binding molecules.

Antibodies of this invention may be monospecific, bispecific or multispecific.

Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen binding domains specific for more than one target polypeptide; see, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. Monospecific anti-MUC16 antibodies or bispecific anti-MUC16/anti-CD3 antibodies of the present invention may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof may be operably linked (e.g., by chemical linkage, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second or additional binding specificity.

The use of the expression anti-CD3 antibody or anti-MUC16 antibody herein is intended to include both monospecific anti-CD3 or anti-MUC16 antibodies, as well as bispecific antibodies containing a CD3 binding arm and a MUC16 binding arm. Thus, the present invention includes bispecific antibodies in which one immunoglobulin arm binds to human CD3 and the other immunoglobulin arm is specific for human MUC16. The CD3 binding arm may contain any of the amino acid sequences HCVR/LCVR or CDR, as indicated in table. 1, 16, 18, 19, 22 and 23 in this document.

In some embodiments, the CD3 binding arm binds to human CD3 and causes activation of human T cells. In some embodiments, the CD3 binding arm weakly binds to human CD3 and induces activation of human T cells. In other embodiments, the CD3 binding arm weakly binds to human CD3 and induces the killing of tumor-associated antigen-expressing cells in the context of a bispecific or multispecific antibody. In other embodiments, the CD3 binding arm binds or weakly binds to human and cynomolgus CD3, but the binding interaction is not detected by in vitro assays known in the art. The MUC16-binding arm may contain any of the amino acid sequences HCVR/LCVR or CDR, as indicated in table. 1 in this document.

According to some illustrative embodiments of the invention, the invention includes bispecific antigen-binding molecules that specifically bind to CD3 and MUC16. Such molecules may be referred to herein as, for example, anti-CD3/anti-MUC16, anti-CD3xMUC16 or CD3xMUC16 bispecific molecules or other similar terminology (eg, anti-MUC16/anti-CD3). The present invention provides bispecific antigen binding molecules designed with a first antigen binding arm that binds to MUC16 and a second antigen binding arm that binds to CD3. In some embodiments, the anti-CD3 arm comprises a heavy chain derived from IGHV3-9*01, IGHJ6*02, IGHD5-12*01. In other embodiments, the bispecific antigen-binding molecule activates human PBMC cells and/or induces cytotoxic activity on tumor antigen-expressing cell lines.

The term MUC16 as used herein refers to the human MUC16 protein unless indicated to be from a non-human species (eg, mouse MUC16, monkey MUC16, etc.). The human MUC16 protein has the amino acid sequence shown in SEQ ID NO: 18 99.

The above-mentioned bispecific antigen binding molecules that specifically bind to CD3 and MUC16 may comprise an anti-CD3 antigen binding molecule that binds to CD3 with weak binding affinity, such as exhibiting a K _D of greater than about 40 nM as measured by an in vitro affinity binding assay. The above bispecific antigen binding molecules may comprise an anti-CD3 antigen binding molecule that binds to CD3 and exhibits an EC50 of greater than about 100 nM as measured by a FACS titration assay. The above bispecific antigen binding molecules may comprise an anti-CD3 antigen binding molecule that does not exhibit measurable or observable binding to CD3 as measured by an in vitro binding affinity assay or FACS titration assay, but retains the ability to activate human PBMC cells and/or induce cytotoxic activity in against tumor antigen-expressing cell lines.

As used herein, the expression antigen binding molecule means a protein, polypeptide, or molecular complex containing or consisting of at least one complementarity determining region (CDR), which alone or in combination with one or more additional CDRs and/or framework regions (FRs) ), specifically associated with a specific

- 25 045730 new antigen. In certain embodiments of the invention, the antigen binding molecule is an antibody or antibody fragment, as those terms are defined elsewhere herein.

As used herein, the expression bispecific antigen binding molecule means a protein, polypeptide, or molecular complex comprising at least a first antigen binding domain and a second antigen binding domain. Each antigen binding domain in a bispecific antigen binding molecule contains at least one CDR that, alone or in combination with one or more additional CDRs and/or FRs, specifically binds to a particular antigen. In the context of the present invention, the first antigen binding domain specifically binds a first antigen (eg, CD3), and the second antigen binding domain specifically binds a second different antigen (eg, MUC16).

In some illustrative embodiments of the present invention, the bispecific antigen binding molecule is a bispecific antibody. Each antigen binding domain of a bispecific antibody contains a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen binding molecule comprising a first and a second antigen binding domain (eg, a bispecific antibody), the CDRs of the first antigen binding domain may be prefixed with A1, and the CDRs of the second antigen binding domain may be prefixed with A2. Thus, the CDRs of the first antigen binding domain may be referred to herein as A1-HCDR1, A1-HCDR2 and A1-HCDR3; and the second antigen binding domain CDRs may be referred to herein as A2-HCDR1, A2-HCDR2, and A2HCDR3.

The first antigen binding domain and the second antigen binding domain can be directly or indirectly linked to each other to form the bispecific antigen binding molecule of the present invention. Alternatively, the first antigen binding domain and the second antigen binding domain may each be associated with a separate multimerization domain. The association of one multimerizing domain with another multimerizing domain facilitates the association between the two antigen binding domains, thereby forming a bispecific antigen binding molecule. As used herein, the term multimerization domain is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to bind to a second multimerization domain of the same or similar structure or constitution. For example, the multimerizing domain may be a polypeptide containing an immunoglobulin C _H 3 domain. A non-limiting example of a multimerizing component is the Fc portion of an immunoglobulin (containing the CH2-CH3 domain), for example, the IgG Fc domain selected from the IgG1, IgG2, IgG3 and IgG4 isotypes, as well as any allotype within each isotype group.

The bispecific antigen binding molecules of the present invention typically contain two multimerizing domains, for example two Fc domains, each of which is individually part of a distinct antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, such as, for example, IgG1/IgG1, IgG2/IgG2, IgG4/IgG4.

Alternatively, the first and second multimerizing domains may be of different IgG isotypes, such as, for example, IgG1/IgG2, IgG1/IgG4, IgG2/IgG4, etc.

In some embodiments, the multimerization domain is an Fc fragment or amino acid sequence ranging from 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the multimerization domain is a cysteine residue or a short cysteine-containing peptide. Other multimerizing domains include peptides or polypeptides containing or consisting of a leucine zipper, a coiled-coil motif, or a coiled-coil motif.

Any bispecific antibody format or technology can be used to create the bispecific antigen binding molecules of this invention. For example, an antibody or fragment thereof having a first antigen-binding specificity may be operably linked (e.g., by chemical binding, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen binding specificity to obtain a bispecific antigen binding molecule. Specific exemplary bispecific formats that may be used in the context of this invention include, but are not limited to, scFv or diabody based bispecific formats, IgG-scFv fusions, double variable domain (DVD)-Ig, Quadroma, peak-to-valley , common light chain (e.g., conventional knob-valley light chain and the like), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgGl/IgG2, Dual Action Fab (DAF)-IgG, and Mab ² (see ., for example, Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein for an overview of the above formats).

In the context of the bispecific antigen binding molecules of the present invention, multimerizing domains, e.g., Fc domains, may include one or more amino acid changes

- 26 045730 (eg, insertions, deletions or substitutions) compared to the wild type, naturally occurring version of the Fc domain. For example, the invention includes bispecific antigen binding molecules containing one or more modifications in the Fc domain, which results in the modified Fc domain having a modified binding interaction (eg, increased or decreased) between the Fc and FcRn. In one embodiment of the invention, the bispecific antigen binding molecule contains a modification at the CH2 or CH3 site, which modification increases the affinity of the Fc domain for FcRn in an acidic environment (for example, in the endosome, where the pH ranges from about 5.5 to about 6.0) . Non-limiting examples of such Fc modifications include, for example, modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (for example, L/Y/F/W or T), 254 (for example, S or T) and 256 (for example, S/R/Q/E/D or T); or modification at position 428 and/or 433 (eg, L/R/S/P/Q or K) and/or 434 (eg, H/F or Y); or modification at position 250 and/or 428; or a modification at position 307 or 308 (eg, 308F, V308F) and 434. In one embodiment of the invention, the modification comprises modification 428L (eg, M428L) and 434S (eg, N434S); modification 428L, 259I (for example, V259I) and 308F (for example, V308F); modification 433K (for example, Н43зК) and 434 (for example, 434Y); modification 252, 254 and 256 (for example, 252Y, 254T and 256e); modification 250Q and 428L (for example, T250Q and M428L); and modification 307 and/or 308 (for example, 308F or з08Р).

This invention also includes bispecific antigen binding molecules comprising a first CH3 domain and a second Ig3 C _H 3 domain, wherein the first and second Ig C _H 3 domains differ from each other by at least one amino acid, and wherein the difference of at least one amino acid reduces binding of a bispecific antibody to protein A compared to a bispecific antibody that has no amino acid difference. 180 In one embodiment of the invention, the first C _H 3 Ig domain binds to protein A, and the second C _H 3 Ig domain contains a mutation that reduces or abolishes binding to protein A, such as modification H95R (as per IMGT exon numbering, H435R as per exon numbering EU). The second _CH 3 may additionally contain the modification Y96F (according to IMGT; Y436F according to the EU); see, for example, US Patent No. 8586713. Other modifications that can be found in the second CH3 include D16E, L18M, N44S, K52N, V57M and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M and V422I according to the EU ) in the case of IgG1 antibodies; N44S, K52N and V82I (according to IMGT; N384S, K392N and V422I according to EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I according to EU) for IgG4 antibodies.

In some embodiments, the Fc domain may be a chimeric one, combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain may comprise part or all of a CH2 sequence derived from the CH2 region of human IgG1, human IgG2, or human IgG4, and part or all of a _CH3 sequence derived from human IgG1, human IgG2, or human IgG4. The chimeric Fc domain may also contain a chimeric hinge region. For example, a chimeric hinge may comprise an upper hinge sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region in combination with a lower hinge sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region. A specific example of a chimeric Fc domain that may be included in any of the antigen-binding molecules set forth herein comprises from N- to C-terminus: [IgG4 C _H 1] - [IgG4 upper hinge] - [IgG2 lower hinge] [IgG4 CH2] - [IgG4 CH3]. Another example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein contains from N- to C-terminus: [IgGl _CH 1] - [upper hinge IgGl] - [lower hinge IgG2] [IgG4 CH2] [IgG1 CH3]. These and other examples of chimeric Fc domains that can be included in any of the antigen binding molecules of this invention are described in US publication 2014/0243504, published August 28, 2014, which is incorporated herein in its entirety. Chimeric Fc domains sharing these common structural arrangements and variants thereof may have altered binding to the Fc receptor, which in turn affects Fc effector function.

In some embodiments, the invention provides an antibody heavy chain wherein the heavy chain constant region (CH) region contains at least 95%, at least 96%, at least 97%, or at least 97% of the amino acid sequence. 98% at least 99% identical to any of SEQ ID NO: 1911, SEQ ID NO: 1912, SEQ ID NO: 1913, SEQ ID NO: 1914, SEQ ID NO: 1915, SEQ ID NO: 1916, SEQ ID NO: 1917, SEQ ID NO: 1918, SEQ ID NO: 1919, or SEQ ID NO: 1920. In some embodiments, the heavy chain constant region (CH) region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1911, SEQ ID NO: 1912, SEQ ID NO: 1913, SEQ ID NO: 1914, SEQ ID NO: 1915, SEQ ID NO: 1916, SEQ ID NO: 1917, SEQ ID NO: 1918, SEQ ID NO: 1919 and SEQ ID NO: 1920.

In other embodiments, the invention provides an antibody heavy chain wherein the Fc domain comprises at least 95%, at least 9-6%, at least 97%, at least 98%, at least 99% identical to

- 27 045730 fight from SEQ ID NO: 1921, SEQ ID NO: 1922, SEQ ID NO: 1923 SEQ ID NO: 1924 SEQ ID NO: 1925, SEQ ID NO: 1926, SEQ ID NO: 1927, SEQ ID NO: 1928 , SEQ ID NO: 1929 or SEQ ID NO: 1930. In some embodiments, the Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1921, SEQ ID NO: 1922, SEQ ID NO: 1923 SEQ ID NO: 1924 SEQ ID NO: 1925, SEQ ID NO: 1926, SEQ ID NO: 1927, SEQ ID NO: 1928, SEQ ID NO: 1929 and SEQ ID NO: 1930.

Sequence options.

The antibodies and bispecific antigen binding molecules of the present invention may contain one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the individual antigen binding domains were derived. . Such mutations can be readily detected by comparing the amino acid sequences described herein with germline sequences available, for example, from publicly available antibody databases. The antigen binding molecules of the present invention may comprise antigen binding domains that are derived from any of the exemplary amino acid sequences described herein, wherein one or more amino acids in one or more framework regions and/or CDR regions are mutated to the corresponding germline sequence residue(s). line from which the antibody was derived, or with the corresponding residue(s) of another human germline sequence, or with a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to collectively herein as germline mutations). One of skill in the art, starting with the heavy and light chain variable region sequences described herein, can readily produce numerous antibodies and antigen binding fragments that contain one or more individual germline mutations or combinations thereof. In some embodiments, all framework residues and/or CDR residues in the VH and/or _VL domains are mutated back to the residues found in the original germline sequence from which the antigen binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only the mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2 or CDR3. In other embodiments of the invention, one or more framework residue(s) and/or CDR residue(s) are mutated with the corresponding residue(s) of another germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antigen-binding domain was originally obtained). In addition, antigen binding domains may contain any combination of two or more germline mutations within framework regions and/or CDR regions, for example, where certain individual residues are mutated to the corresponding residue of a certain germline sequence, while certain other residues that differ from the original germline sequence are conserved or mutate with the corresponding residue of another germline sequence. Once produced, antigen binding domains that contain one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding or binding affinity, improved or enhanced antagonistic or agonistic biological properties (as appropriate ), reduced immunogenicity, etc. Bispecific antigen binding molecules containing one or more antigen binding domains prepared in this general manner are covered by this invention.

The invention also includes antigen binding molecules, wherein one or both antigen binding domains comprise variants of any of the HCVR, LCVR and/or CDR amino acid sequences described herein having one or more conservative substitutions. For example, the present invention includes antigen binding molecules comprising an antigen binding domain having the amino acid sequences HCVR, LCVR and/or CDR with, for example, 10 or less, 8 or less, 6 or less, 4 or less, and the like of conservative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences described herein. A conservative amino acid substitution is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution will not significantly change the functional properties of the protein. Examples of groups of amino acids with side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) main side chains: lysine, arginine and histidine; 6) acidic side chains: aspartate and glutamate and 7) sulfur dioxide

- 28 045730 compressing side chains - cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix described in Gonnet et al. (1992) Science 256: 1443-1445, incorporated herein by reference. A moderately conservative replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

The invention also includes antigen binding molecules comprising an antigen binding domain with an HCVR, LCVR, and/or CDR amino acid sequence that is substantially identical to any of the HCVR, LCVR and/or CDR amino acid sequences described herein. The term substantial identity or substantially identical, when referring to an amino acid sequence, means that two amino acid sequences, when optimally aligned, for example using the GAP or BESTFIT programs using default gap weights, share at least 95% identity sequence, even more preferably at least 98% or 99% sequence identity. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage sequence identity or degree of similarity can be adjusted upward to correct the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art; see, for example, Pearson (1994) Methods Mol. Biol. 24:3 07-331, incorporated herein by reference.

Sequence similarity for polypeptides, also called sequence identity, is typically measured using sequence analysis software. Protein analysis software finds similar sequences using similarity measurements assigned to various substitutions, deletions and other modifications, including conserved amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and its mutein; see for example GCG version 6.1. Polypeptide sequences can also be compared using FASTA with standard or recommended parameters; program in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignment identity and sequence percentage of regions of best overlap between the query sequence and the search sequence (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of this invention to a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN, using default parameters; see, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-402, each of which is incorporated herein by reference.

pH-dependent binding.

The present invention includes anti-MUC16 antibodies and anti-CD3/anti-MUC16 bispecific antigen-binding molecules, with pH-dependent binding characteristics. For example, the anti-MUC16 antibody of the present invention may exhibit reduced binding to MUC16 at acidic pH compared to neutral pH. Alternatively, anti-MUC16 antibodies of the present invention may exhibit enhanced binding to MUC16 at acidic pH compared to neutral pH. The expression acidic pH includes pH values less than about 6.2, such as about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5. 6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or less. As used herein, the expression neutral pH means a pH of about 7.0 to about 7.4. The expression neutral pH includes pH values around 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

In some cases, reduced binding...at acidic pH compared to neutral pH is expressed in terms of the ratio of the KD value of an antibody binding to its antigen at acidic pH to the KD value of an antibody binding to its antigen at neutral pH (or vice versa). For example, an antibody or antigen binding fragment thereof may be considered to exhibit reduced binding to MUC16 at acidic pH compared to neutral pH for purposes of the present invention if the antibody or antigen binding fragment thereof has an acidic/neutral KD ratio of about 3.0 or greater. In some exemplary embodiments, the acidic/neutral KD for an antibody or antigen binding fragment of the invention may be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 , 6.5, 7.0, 7.5, 8.0, 8.5 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12, 5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or more.

Antibodies with pH-dependent binding characteristics can be obtained, for example, by screening a population of antibodies to reduce (or enhance) binding to a particular antigen at

- 29 045730 acidic pH, compared to neutral pH. In addition, modifications of the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent characteristics. For example, by replacing one or more amino acids of the antigen binding domain (eg, in the CDR) with a histidine residue, an antibody with reduced antigen binding at an acidic pH relative to a neutral pH can be obtained.

Antibodies containing Fc variants.

In accordance with some embodiments of the present invention, anti-MUC16 antibodies, and anti-CD3/anti-MUC16 bispecific antigen-binding molecules, which are provided to contain an Fc domain containing one or more mutations that increase or decrease binding of the antibody to the receptor FcRn, for example, at acidic pH compared to neutral pH. For example, the present invention includes antibodies containing a mutation in the C _H 2 or C _H 3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain for FcRn in an acidic environment (for example, in the endosome, where the pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in the half-life of the serum antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (for example, L/Y/F/W or T), 254 (for example, S or T) and 256 (for example, S/R/Q/E/D or T); or modification at position 428 and/or 433 (eg, H/L/R/S/P/Q or K) and/or 434 (eg, H/F or Y); or modification at position 250 and/or 428; or a modification at positions 307 or 308 (eg, 308F, V308F) and 434. In one embodiment of the invention, the modification comprises modification 428L (eg, M428L) and 434S (eg, N434S); modification 428L, 259I (for example, V259I) and 308F (for example, V308F); modification 433K (for example, Н433К) and 434 (for example, 434Y); modification 252, 254 and 256 (for example, 252Y, 254T and 256E); modification 250Q and 428L (for example, T250Q and M428L); and modification 307 and/or 308 (for example, 308F or 308P).

For example, the present invention includes anti-MUC16 antibodies and anti-CD3/anti-MUC16 bispecific antigen-binding molecules containing an Fc domain containing one or more mutation pairs or groups selected from the group consisting of: 250Q and 248L (e.g. T250Q and M248L); 252Y, 254T and 256E (for example, M252Y, S254T and T256E); 428L and 434S (for example, M428L and N434S); and 433K and 434F (for example, Н433К and N434F). All possible combinations of the above Fc domain mutations and other mutations in the variable domains of the antibodies described herein are contemplated within the scope of this invention.

Biological characteristics of antibodies and bispecific antigen-binding molecules.

This invention includes antibodies and antigen-binding fragments thereof that bind to human MUC16 with high affinity (eg, subnanomolar KD values).

In accordance with some embodiments of the invention, the invention includes antibodies and antigen-binding antibody fragments that bind to human MUC16 (e.g., at 25° C.) with a KD of less than about 60 nM, as measured by surface plasmon resonance, e.g. using the analysis format as defined in Example 4 herein. In some embodiments, antibodies or antigen binding fragments of the invention bind to MUC16 with a KD of less than about 60 nM, less than about 40 nM, less than about 20 nM, less than about 10 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 800 pM, less than about 700 pM, less than about 500 pM, less than about 400 pM, or less than about 300 pM, as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 4 herein (e.g., mAb capture or capture format antigen), or a substantially similar assay. This invention includes bispecific antigen binding molecules (e.g., bispecific antibodies that bind to MUC16 with a KD of less than about 7 nM, as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 4 herein (e.g., capture format mAb or antigen capture), or a substantially similar assay.

The invention also includes antibodies and antigen-binding fragments that bind to MUC16 with a dissociating half-life (t _1/2 ) of greater than about 10 minutes or greater than about 125 minutes, as measured by surface plasmon resonance at 25°C, for example , using an analysis format as defined in Example 4 herein, or a substantially similar analysis. In some embodiments, antibodies or antigen binding fragments of the invention bind to MUC16 with a t ^1/2 greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 110 minutes, or greater than about 120 minutes, measured using surface plasmon resonance at 25 °C, for example, using an assay format as defined in Example 4 herein (eg, mAb capture or antigen capture format), or a substantially similar assay. This invention includes bispecific antigen binding molecules (e.g., bispecific antibodies that bind to MUC16 with more than

- 30 045730 about 10 minutes or more than about 20 minutes, as measured by surface plasmon resonance at 25°C, for example, using the assay format as defined in Example 4 herein, or a substantially similar assay.

The invention also includes antibodies and antigen-binding fragments that specifically bind to human cell lines that express endogenous MUC16 (eg, OVCAR-3), as determined by an electrochemiluminescence detection assay as described in Example 2, or a substantially similar assay .

The present invention also includes anti-CD3/anti-MUC16 bispecific antigen binding molecules that have one or more characteristics selected from the group consisting of: (a) inhibiting tumor growth in immunocompromised mice bearing human ovarian cancer xenografts; and (b) inhibiting tumor growth of established tumors in immunocompromised mice bearing human ovarian cancer xenografts (see, for example, Example 8).

This invention includes antibodies and antigen-binding fragments thereof that bind to human CD3 with high affinity. The invention also includes antibodies and antigen-binding fragments thereof that bind to human CD3 with moderate or low affinity, depending on the therapeutic context and the specific target properties desired. In some cases, low affinity includes antibodies that bind to CD3 with a KD or EC ₅₀ (eg, as measured in a surface plasmon resonance assay) of greater than 300 nM, greater than 500 nM, or greater than 1 μM. The invention also includes antibodies and antigen-binding fragments thereof that bind to human CD3 without measurable affinity. For example, in the context of a bispecific antigen-binding molecule in which one arm binds to CD3 and the other arm binds to a target antigen (e.g., MUC16), it may be desirable that the target antigen-binding arm binds to the target antigen with high affinity while anti-CD3 The arm binds to CD3 only with moderate or low affinity or no affinity. In this way, preferential targeting of the antigen-binding molecule to cells expressing the target antigen can be achieved while avoiding common/off-target CD3 binding and associated side effects.

The present invention includes bispecific antigen binding molecules (eg, bispecific antibodies) that are capable of simultaneously binding to human CD3 and human MUC16. The binding arm that interacts with cells that express CD3 may have weak or undetectable binding as measured in a suitable in vitro binding assay. The extent to which a bispecific antigen binding molecule binds cells that express CD3 and/or MUC16 can be assessed using fluorescence-activated cell sorting (FACS), as shown in Example 5 herein.

For example, the invention includes antibodies, antigen binding fragments, and bispecific antibodies thereof that specifically bind human T cell lines that express CD3 but do not express MUC16 (e.g., Jurkat), primate T cells (e.g., peripheral blood mononuclear cells cynomolgus monkey [PBMC]) and/or cells expressing MUC16.

The present invention includes antibodies, antigen binding fragments, and bispecific antibodies thereof that bind to human CD3 with weak (ie, low) or even no detectable binding or binding affinity.

The present invention includes antibodies, antigen binding fragments, and bispecific antibodies thereof that bind to monkey (ie, cynomolgus) CD3 with weak (ie, low) or even no detectable binding or binding affinity.

This invention includes antibodies, antigen binding fragments, and bispecific antibodies thereof that bind to human CD3 and induce T cell activation.

The present invention includes anti-CD3/anti-MUC16 bispecific antigen-binding molecules that are capable of destroying a tumor antigen-expressing cell in a subject (see, for example, Example 8, in bioluminescence imaging analysis or a substantially similar assay). For example, in accordance with some embodiments of the invention, anti-CD3/anti-MUC16 bispecific antigen binding molecules are provided, wherein a single administration of 10 μg of the bispecific antigen binding molecule to a subject causes a decrease in the number of MUC16-expressing cells in the subject (e.g., tumor growth in the subject is inhibited or inhibited). Unless otherwise stated, bioluminescent emission refers to [f/s/cm ² /sr].

The present invention also includes anti-MUC16 antibody-drug conjugates that inhibit tumor growth in in vivo MUC16-positive ovarian cancer xenograft models (see, for example, Example 10, in a bioluminescence imaging assay or substantially similar assay). In some embodiments, provided are anti-MUC16 antibody-drug conjugates with Compound 7 in which four once-weekly doses administered at 85 μg/kg inhibit intraperitoneal OVCAR3/luc tumor growth in vivo. In some embodiments of the invention, anti-MUC16 antibody-drug conjugates with Compound 7 are provided, in which four once-weekly doses administered at a dose of 85

- 31 045730 µg/kg, inhibit subcutaneous OVCAR3/luc tumor growth in vivo. In some embodiments, provided are anti-MUC16 antibody-drug conjugates with Compound 10, wherein a single dose of 85 μg/kg, 170 μg/kg, or 340 μg/kg inhibits intraperitoneal OVCAR3/luc tumor growth in vivo. Unless otherwise stated, bioluminescent emission refers to [f/s/cm ² /sr].

The present invention also includes anti-CD3/anti-MUC16 bispecific antigen-binding molecules that exhibit pharmacokinetic profiles in humanized MUC16 x CD3 mice (mice homozygous for human MUC16 and CD3 expression, MUC16 ^hu/hu x CD3 ^hu/hu ) humanized CD3 mice ( mice homozygous for human CD3 expression, CD3 ^hu / ^hu ) and strain-matched wild-type (WT) mice (75% C57BL, 25% 129Sv) as described in Example 7 and shown in FIG. 1, 2 and 3.

Epitope mapping and related technologies.

The epitope on CD3 and/or MUC16 to which the antigen-binding molecules of this invention bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of CD3 or MUC16 protein. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) of CD3 or MUC16. Antibodies of the present invention may react with amino acids contained on one CD3 chain (eg, CD3 epsilon, CD3 delta, or CD3 gamma) or may react with amino acids on two or more different CD3 chains. As used herein, the term epitope refers to an antigenic determinant that interacts with a specific antigen-binding site on a variable region of an antibody molecule known as a paratope. One antigen can have more than one epitope. Thus, different antibodies may bind to different regions on the antigen and may have different biological effects. Epitopes can be conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, the epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

Various methods known to those skilled in the art can be used to determine whether the antigen binding domain of an antibody interacts with one or more amino acids within a polypeptide or protein.

Exemplary methods include, for example, routine cross-blocking assays such as those described by Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scan mutation assays, peptide blot assays (Reineke, 2004, Methods Mol Biol 248:443–463) and peptide cleavage assay. In addition, methods such as epitope removal, epitope extraction, and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify amino acids within a polypeptide with which the antigen binding domain of an antibody interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general, the hydrogen/deuterium exchange method involves labeling a protein of interest with deuterium, followed by binding of an antibody to the deuterium-labeled protein. The protein/antibody complex is then transferred to water to allow hydrogen-deuterium exchange across all residues except the antibody-protected residues (which remain deuterium-labeled). Once the antibody is dissociated, the target protein is subjected to protease digestion and analysis by mass spectrometry, thereby identifying deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts; see, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 13: 256A265A. X-ray crystallography of the antigen/antibody complex can also be used for epitope mapping.

The invention further includes anti-MUC16 antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (e.g., antibodies containing any of the amino acid sequences set forth in Table 1 herein) . Likewise, the present invention also includes anti-MUC16 antibodies that compete for binding to MUC16 with any of the specific exemplary antibodies described herein (e.g., antibodies containing any of the amino acid sequences as set forth in Table 1 herein) .

The invention also includes bispecific antigen binding molecules comprising a first antigen binding domain that specifically binds to human CD3 and/or cynomolgus CD3 with low or no binding or binding affinity, and a second antigen binding domain that specifically binds to human MUC16, wherein the first antigen binding domain domain binds to the same epitope on CD3 as any of the specific exemplary CD3-specific antigen binding domains described herein and/or wherein the second antigen binding domain binds to the same epitope on MUC16 as any of

- 32045730 specific illustrative MiC16-specific antigen binding domains described herein.

In addition, the present invention also includes bispecific antigen binding molecules comprising a first antigen binding domain that specifically binds to human CD3 and a second antigen binding domain that specifically binds to human MUC16, wherein the first antigen binding domain competes for binding to CD3 with any of the specific exemplary CD3-specific antigen binding domains described herein, and/or wherein the second antigen binding domain competes for binding to MUC16 with any of the specific exemplary MUC16-specific antigen binding domains described herein.

Whether a particular antigen-binding molecule (eg, an antibody) or its antigen-binding domain binds to the same epitope or competes for binding with a reference antigen-binding molecule of the present invention can be readily determined using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope on MUC16 (or CD3) as a reference bispecific antigen binding molecule of the present invention, the reference bispecific molecule is first allowed to bind to the MUC16 protein (or CD3 protein). The ability of the test antibody to bind to the MUC16 (or CD3) molecule is then assessed. If the test antibody is able to bind to MUC16 (or CD3) after saturation binding to the reference bispecific antigen binding molecule, it can be concluded that the test antibody binds to a different epitope of MUC16 (or CD3) than the reference bispecific antigen binding molecule. On the other hand, if the test antibody is unable to bind to a MUC16 (or CD3) molecule after saturation binding to a reference bispecific antigen binding molecule, then the test antibody may bind to the same MUC16 (or CD3) epitope as the epitope bound by the reference bispecific antigen binding molecule this invention. Additional routine testing (eg, peptide mutation and binding assays) can then be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference bispecific antigen-binding molecule, or whether a steric blockage (or other phenomenon) responsible for the lack of observed binding. Experiments of this type can be performed using ELISA, RIA (radioimmunoassay), Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. In accordance with some embodiments of the present invention, two antigen binding proteins bind to the same (or overlapping) epitope if, for example, a 1-, 5-, 10-, 20-, or 100-fold excess of one antigen binding protein inhibits the binding of the other by at least 50%, but preferably 75%, 90% or even 99%, as measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res. 1990: 50: 1495-1502). Alternatively, two antigen binding proteins are considered to bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antigen binding protein reduce or eliminate the binding of the other. Two antigen binding proteins are considered to have overlapping epitopes if only a subset of amino acid mutations that reduce or eliminate the binding of one antigen binding protein reduce or eliminate the binding of the other.

To determine whether an antibody or its antigen-binding domain competes for binding to a reference antigen-binding molecule, the binding technique described above is performed in two orientations: in the first orientation, the reference antigen-binding molecule is allowed to bind the MUC16 protein (or CD3 protein) under saturation conditions, followed by an assessment of the binding of the test antibody with the MUC16 (or CD3) molecule. In the second orientation, the test antibody is allowed to bind to the MUC16 (or CD3) molecule under saturated conditions, followed by assessment of the binding of the reference antigen binding molecule to the MUC16 (or CD3) molecule. If in both orientations only the first (saturating) antigen binding molecule is able to bind to the MUC16 (or CD3) molecule, then it is concluded that the test antibody and the reference antigen binding molecule are competing for binding to MUC16 (or CD3). As will be appreciated by one skilled in the art, an antibody that competes for binding to a reference antigen binding molecule may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope.

Preparation of antigen-binding domains and construction of bispecific molecules.

Antigen-binding domains specific for particular antigens can be generated using any antibody generation technology known in the art. Once prepared, two different antigen binding domains specific for two different antigens (eg, CD3 and MUC16) can be suitably positioned relative to each other to produce a bispecific antigen binding molecule of the present invention using routine methods. (Discussion of illustrative bispecific antibody formats that can be

- 33 045730 used for the construction of bispecific antigen binding molecules of this invention, is provided elsewhere in this document). In certain embodiments of the invention, one or more individual components (eg, heavy and light chains) of the multispecific antigen binding molecules of the present invention are derived from chimeric, humanized, or fully human antibodies. Methods for producing such antibodies are well known in the art. For example, one or more heavy and/or light chains of the bispecific antigen binding molecules of the present invention can be produced using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other technology that generates human antibodies), chimeric antibodies with high affinity for a specific antigen (eg CD3 or MUC16) are initially isolated having a human variable region and a mouse constant region. Antibodies are characterized and selected for desired characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the desired human constant region to generate fully human heavy and/or light chains that can be incorporated into the bispecific antigen binding molecules of the present invention.

Genetically engineered animals can be used to produce human bispecific antigen-binding molecules. For example, a genetically modified mouse may be used that is unable to reorganize and express the endogenous mouse immunoglobulin light chain variable sequence, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to the mouse kappa constant gene in the endogenous mouse kappa locus. Such genetically modified mice can be used to produce fully human bispecific antigen binding molecules containing two different heavy chains that are linked to an identical light chain that contains a variable domain derived from one of two different human light chain variable region gene segments. (See for example US 2011/0195454). The term fully human refers to an antibody or antigen binding fragment or immunoglobulin domain thereof comprising an amino acid sequence encoded by DNA derived from a human sequence throughout the entire length of each polypeptide of the antibody or antigen binding fragment or immunoglobulin domain thereof. In some cases, the entirely human sequence is derived from a protein endogenous to humans. In other cases, the entirely human protein or protein sequence comprises a chimeric sequence, each component sequence being derived from a human sequence. Without being bound by any particular theory, chimeric proteins or chimeric sequences are generally designed to minimize the formation of immunogenic epitopes at component sequence junctions, for example, compared to any regions or domains of wild-type human immunoglobulin.

Bioequivalents.

The present invention covers antigen binding molecules having amino acid sequences that differ from those of the exemplary molecules described herein, but which retain the ability to bind CD3 and/or MUC16. Such variant molecules may contain one or more insertions, deletions or substitutions of amino acids from the original sequence, but exhibit biological activity that is substantially equivalent to that of the described bispecific antigen binding molecules.

The present invention includes antigen binding molecules that are bioequivalent to any of the exemplary antigen binding molecules described herein. Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, whether in a single dose or multiple doses. dose. Some antigen-binding proteins will be considered equivalents or pharmaceutical alternatives if they are equivalent in their extent of absorption, but not in their rate of absorption, and may still be considered bioequivalent because such differences in absorption rate are intentional and reflected in the labeling and are not essential to achieving effective concentrations of the drug, for example, for chronic use, and are considered medically insignificant for the particular drug product being studied.

In one embodiment of the present invention, two antigen binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity and potency.

In one embodiment of the invention, two antigen binding proteins are bioequivalent if the patient can switch one or more times between the reference drug and the biologic without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity or reduced efficacy compared with pro

- 34 045730 ongoing therapy without such a switch.

In one embodiment of the invention, two antigen binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent such mechanisms are known.

Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) an in vivo test in humans or other mammals in which the concentration of an antibody or its metabolites is measured in blood, plasma, serum or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and reliably predicts in vivo human bioavailability data; (c) an in vivo test in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical study that establishes the safety, efficacy or bioavailability or bioequivalence of the antigen binding protein.

Bioequivalent versions of the illustrative bispecific antigen binding molecules set forth herein can be constructed, for example, by making various substitutions of residues or sequences or removing terminal or internal residues or sequences not required for biological activity. For example, cysteine residues that are not essential for biological activity can be removed or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antigen binding proteins may include variants of the illustrative bispecific antigen binding molecules set forth herein containing amino acid substitutions that modify the glycosylation characteristics of the molecules, for example, mutations that eliminate or remove glycosylation.

Species Selectivity and Species Cross-Reactivity In accordance with some embodiments of the invention, antigen binding molecules are provided that bind to human CD3 but not to CD3 from other species. Also provided are antigen binding molecules that bind to human MUC16. The invention also includes antigen-binding molecules that bind to human CD3 and to CD3 of one or more non-human species; and/or antigen-binding molecules that bind to human MUC16.

In accordance with some illustrative embodiments of the invention, antigen binding molecules are provided that bind to human CD3 and/or human MUC16 and may or may not bind, as appropriate, to one or more mouse, rat, guinea pig CD3 and/or MUC16 , hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus, monkey, rhesus or chimpanzee. For example, a specific embodiment of the present invention provides bispecific antigen binding molecules comprising a first antigen binding domain that binds human CD3 and cynomolgus CD3, and a second antigen binding domain that specifically binds human MUC16.

Antibody-drug conjugates (ADCs).

This invention provides antibody-drug conjugates (ADCs) containing an anti-MUC16 antibody or antigen-binding fragment thereof conjugated to a therapeutic molecule, such as a cytotoxic agent, a chemotherapeutic drug, an immunosuppressant, or a radioisotope. In general terms, an ADC contains: A - [L - P] _y , where A is an antigen binding molecule, for example, an anti-MUC16 antibody or fragment thereof (for example, a fragment containing at least HCDR3 selected from any of the amino acid sequences of HCDR3 listed in Table 1), L is a linker, P is a payload or therapeutic moiety (eg, a cytotoxic agent), and y is an integer from 1 to 30. In various embodiments, the ADC comprises anti-MUC16 an antibody or antigen-binding fragment thereof that contains the HCVR and LCVR CDRs having the amino acid sequences of SEQ ID NO (eg, SEQ ID NO: 2 and 10) listed in table. 1, or specific HCVR/LCVR pairs (eg, SEQ ID NO: 2/10). In some cases, the anti-MUC16 antibody or fragment thereof contains a CDR with the amino acid sequences SEQ ID NO (for example, SEQ ID NO: 4-68-12-14-16) shown in table. 1. In some cases, the anti-MUC16 antibody or fragment thereof contains HCVR and LCVR having the amino acid sequences of SEQ ID NO (for example, SEQ ID NO: 2 and 10) presented in table. 1, or pairs of specific amino acid sequences (eg, SEQ ID NO: 2/10). In some cases, an anti-MUC16 antibody is an antibody or antigen binding fragment that binds to human MUC16 at one or more of the five membrane-proximal human MUC16 SEA domains corresponding to residues 13791-14451 of SEQ ID NO: 1899. In some cases, anti-MUC16 an antibody is an antibody or antigen binding fragment that binds to human MUC16 at residues 13810-14451 of SEQ ID NO: 1899. In some cases, an anti-MUC16 antibody is an antibody or antigen binding fragment that binds to any of several SEA1, SEA2, SEA3, SEA4, SEA5, SEA6, SEA7, SEA8,

- 35 045730

Human SEA9, SEA10, SEA11, SEA12, SEA13, SEA14, SEA15 or SEA16 MUC16.

Cytotoxic agents include any agent that is harmful to the growth, viability or reproduction of cells. The antigen-binding molecules or antibodies of this invention deliver these cytotoxic agents, referred to herein as payloads, to target cells. Examples of suitable cytotoxic agents and chemotherapeutic agents for ADC formation are known in the art.

Examples of suitable cytotoxic agents and chemotherapeutic agents that can be conjugated to anti-MUC16 antibodies in accordance with this aspect of the present invention include, for example, 1-(2-chloroethyl)-1,2 dimethanesulfonylhydrazide, 1,8-dihydroxybicyclo[7.3. 1]trideca-4,9-dien-2,6-dien-13-one, 1-dehydrotestosterone, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, 9-amine-camptothecin, actinomycin D, amanitins, aminopterin, anguidine , anthracycline, anthramycin (AMC), auristatins (monomethyl auristatin E or monomethyl auristatin F), bleomycin, bisulfan, butyric acid, calicheamicins, camptothecin, carminomycin, carmustine, kemadotins, cisplatin, colchicine, combretastatins, cyclophosphamide, cytarabine, cytochalasin B, dactinomycin , daunorubicin, decarbazine, diacetoxypentyldoxorubicin, dibromomannitol, dihydroxy anthracine dione, disorazoles, dolastatin, doxorubicin, duocarmycin, echinomycins, eleutherobines, emetine, epothilones, esperamycin, estramustines, ethidium bromide, etoposide, fluorouracils, geldanamycins, gram icidin D, glucocorticoids, irinotecans, leptomycins , leurosines, lidocaine, lomustine (CCNU), maytansinoids, mechlorethamine, melphalan, mercatopurines, methopterins, methotrexate, mithramycin, mitomycin, mitoxantrone, N8-acetyl spermidine, podophyllotoxins, procaine, propranolol, pteridines, pyromycin, rhizoxins, streptozotocin, ta lisomycins, taxol , tenoposide, tetracaine, thioepa chlorambucil, tomaimycins, topotecans, tubulisin, vinblastine, vincristine, vindesine, vinorelbine, and derivatives of any of the above.

In accordance with some embodiments of the invention, the cytotoxic agent that is conjugated to the anti-MUC16 antibody is an auristatin, such as monomethyl Auristatin E (MMAE) or monomethyl Auristatin F (MMAF), a tubulisin, such as TUB-OH or TUB-OMOM, a derivative tomaimycin, dolastatin or maytansinoid derivatives such as DM1 or DM4. In some embodiments, the cytotoxic agent is a maytansinoid having the structure of formula (I), including stereoisomers of compounds of formula (I):

O CH ₃ (Formula I) where A represents arylene or heteroarylene.

In some embodiments, A is a divalent benzene, pyridine, naphthalene or quinolone radical, which is optionally substituted.

In some embodiments, A is arylene.

In some embodiments of the invention, A is:

where R ¹ independently represents in each case alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl, halogen, heteroaryl, heterocycloalkyl, hydroxyl, cyano, nitro,

or azido, where RA represents alkyl or heteroalkyl;

n is an integer from 0 to 4;

m is an integer from 0 to 3;

p is an integer from 0 to 6; and q is an integer from 0 to 5.

- 36 045730

In some embodiments of the present invention, the compound of Formula I is selected from the group consisting of:

- 37 045730

- 38 045730

- 39 045730

In some embodiments, the maytansinoid of formula (I) is conjugated to an anti-MUC16 antibody or antigen binding moiety via a linker as shown in formula (IA) below:

(Formula IA) wherein A is arylene or heteroarylene, as discussed above in connection with formula (I); L represents a linker;

BA is an anti-MUC16 antibody or antigen-binding fragment thereof;

k is an integer between 1 and 30.

In various embodiments of the invention, L is:

A

4-8Ρ-ΑΑ ¹ -ΑΑ ² ψ where SP represents a spacer;

represents one or more links to an anti-MUC16 antibody or fragment thereof;

AA ¹ is an amino acid; and AA ² is an amino acid.

In some embodiments, AA1-AA ² is: valinecitrulline, citrulline-valine, lysine-phenylalanine, phenylalanine-lysine, valine-asparagine, asparagine-valine, threonine-asparagine, asparagine-threonine, serine-asparagine, asparagine-serine, phenylalanine asparagine , asparagine-phenylalanine, leucine-asparagine, asparagine-leucine, isoleucine-asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acid-asparagine, asparagine glutamic acid, citrulline-asparagine, asparagine-citrulline, alanine-asparagine or asparaginalanine.

In some embodiments of the invention, SP is:

N ^S

N~(CH ₂ ) _b -N— or

where is a link with an anti-MUC16 antibody or fragment thereof; b represents an integer between 2 and 8.

In other embodiments of the invention, L is:

where is a link with an anti-MUC16 antibody or fragment thereof; b represents an integer between 2 and 8.

- 40 045730

In one embodiment of the invention, the compound of formula (IA), including a linker that binds to an anti-MUC16 antibody or antigen binding fragment thereof, is:

where ⁵ represents a linkage to an anti-MUC16 antibody or fragment thereof. In some cases this fragment is called Compound 10.

In one embodiment of the invention, the compound of formula (IA), including a linker that binds to an anti-MUC16 antibody or antigen binding fragment thereof, is:

where * represents a linkage to an anti-MUC16 antibody or fragment thereof. In some cases this fragment is called Compound 60.

In some embodiments, the cytotoxic agent is a maytansinoid having the structure of formula (II), including stereoisomers of compounds of formula (II):

° сн ₃ (Formula II) where Asa is an amino acid, a peptide having 2-20 amino acids, alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, -CR5R6-, -O-, -C(=O)- , -О-С(=О)-, -С(=О)-О-, -О-С(=О)-О-, -С(=О)-(SI _Х ) _Р 1-, -С (=O)-O-(SI _X ) _R 1-, -(CH _X ) _R 1-C(=O)-, -(CHx) _R 1-C(=O)-O-, -(O- (CH ₂ )p ₂ -)p ₃ -, ((CH ₂ )p ₂ -O-)p ₃ -, -C(=S)-, -C(=S)-S-, -C(=S )-NH-, -SC(=S)-, -SC(=S)-S-, -S-, -SO-, -SO2-, -NR4-, -N(R4)-C(=O) -N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -С(=O)-N(R ₄ )-, -C (=O)-N(R4)-C(=O)- or -OC(=O)-NR4-, where alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are optionally substituted; and pl, p2 and p3 are each independently equal to 0 or an integer from 1 to 100;

x is 0, 1 or 2;

R ₄ , R ₅ , R ₆ and R ₈ are each independently H, either substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl or heterocyclyl; And

R _4a is substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl or heterocyclyl.

- 41 045730

In some embodiments of the present invention, the compound of formula (II) is selected from the group consisting of:

In one embodiment of the invention, the compound of formula (II) is:

In some embodiments, the maytansinoid of formula (II) is conjugated to an anti-MUC16 antibody or antigen binding moiety via a linker as shown in formula (IIA) below:

(Formula HA) wherein BA is an anti-MUC16 antibody or an antigen-binding fragment thereof;

a is an integer from 1 to 30;

Z2 is the following structural formula: -Z2A-Z2B-Z2C-Z2D, wherein each _Z2A , Z2B, _Z2C and Z2D are independently absent, amino acid, peptide having 2-20 amino acids, alkyl, alkynyl, alkenyl, cycloalkyl, aryl , heteroaryl, heterocyclyl, -CR5R6-, -O-, -C(=O)-, -O-C(=O)-, -C(=O)-O-, -O-C(=O)- О-, -С(=О)-(СН _x )р1, -C(=O)-O-(CHx)pi, -(СНх)р1-С(=О)-, -(СН _x )р1- C(=O)-O-, -(O-(CH2)p2-)p3-, -((CH2)p2-O-)p3-, -C(=S)-, -C(=S)- S-, -C(=S)-NH-, -SC(=S)-, -SC(=S)-S-, -S-, -SO-, -SO2-, -NR4-, -N( R4)-C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N (R4)-, -C(=O)-N(R4)-C(=O)-, -OC(=O)-N(R4), -OC(=S)-N(R4)-, - C(=S)-N(R4)-, -N=C=S, -N=C=O,

- 42 045730

A is a natural or unnatural amino acid or peptide containing 2-20 amino acids;

W represents -O-, -S-, -CR5R6- or -NR4-;

X represents aryl, heteroaryl, cycloalkyl or heterocyclyl, where aryl, heteroaryl, cycloalkyl and heterocyclyl are optionally substituted;

where A1, A ₃ and R1, each independently, represent an amino acid, a peptide having 2-20 amino acids, alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, -CR ₅ R ₆ -, -O-, -C (=O)-, -O-C(=O)-, -C(=O)-O-, -O-C(=O)-O-, -C(=O)-(CH _x )р1 -, -C(=O)-O-(CH _x )p1-, -(CH _x )p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O -(CH2)р2-)р3-, -((СН2)р2-О-)р3-, -C(=S)-, -C(=S)-S-, -SC(=S)-, - C(=S)-NH-, -SC(=S)-S-, -S-, -so-, -SO2-, -NR4-, -N(R4)-C(=O)-N(R8 )-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, -C(=O)- N(R ₄ )-C(=O) - or -OC(=O)-NR4-, where alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are optionally substituted;

R ₁₇ is selected from the group consisting of O, S, NR18 and CR5R6;

R18 is selected from the group consisting of H, alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl and acyl, where alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl and acyl are optionally substituted;

R4, R5, R6 and R8 are each independently H, either substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl or heterocyclyl;

R _4a is substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl or heterocyclyl;

p1, p2 and p3 are each independently equal to 0 or an integer from 1 to 100; and x is 0, 1 or 2.

In some embodiments of formula (IIA), A is a peptide selected from the group consisting of valine-citrulline, citrulline-valine, lysine-phenylalanine, phenylalanine-lysine, valine-asparagine, asparagine-valine, threonine-asparagine, asparagine-threonine , serineasparagine, asparagine-serine, phenylalanine-asparagine, asparagine-phenylalanine, leucineasparagine, asparagine-leucine, isoleucine-asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acid-asparagine, asparagine-glutamic acid, citrulline asparagine, asparagine -citrulline, alanine-asparagine and asparagine-alanine.

In one embodiment of the invention, a compound of formula (IIA) that binds to an anti-MUC16 antibody or antigen binding fragment is as follows:

where " represents a linkage to an anti-MUC16 antibody or fragment thereof. In some cases this fragment is called Compound 7.

In some embodiments, the cytotoxic agent that is conjugated to the anti-MUC16 antibody or fragment thereof is a pure, or substantially pure, diastereomer of DM1:

o sn ₃ (DM1) and y is an integer from 1 to 0.

In another embodiment of the invention, the ADC contains _an A - [L - P] structure, in which A is an anti-MUC16 antibody or antigen binding fragment thereof, and [L - P] is:

- 43 045730 about сН ₃ or a mixture thereof, and where y represents an integer from 1 to 30, and “ represents a bond with an anti-MiC16 antibody or a fragment thereof.

Other maytansinoid derivatives are described in WO 2014/145090, WO2016/160615, and WO 2015/031396, each of which is incorporated herein by reference in its entirety.

In some embodiments, the cytotoxic agent that is conjugated to the anti-MUC16 antibody or fragment thereof is MMAEM or MMAF.

Other cytotoxic agents known in the art are within the scope of this invention, including, for example, protein toxins such as ricin, C. difficile toxin, Pseudomonas exotoxin, diphtheria toxin, botulinum toxin, briodine, saporin, lactox toxins (the there are phytolaccatoxin and phytolacigenin) and others, such as those described in Sapra et al., Pharmacol. & Therapeutics, 2013, 138: 452-469.

Cytotoxic agents (payloads) can be linked to the anti-MUC16 antigen-binding molecule or antibody of the present invention through a chemical linker that covalently binds the payload compound to the protein molecule (ie, antibody). Exemplary embodiments of specific linkers are discussed above. More generally, and as used herein, the term linker refers to any divalent group or moiety that binds, connects, or binds a binding agent (eg, an antibody or antigen-binding fragment thereof) to a specified payload compound herein. In general, suitable binding agent linkers for the antibody conjugates described herein are those that are stable enough to utilize the circulating half-life of the antibody while still being capable of releasing its payload following antigen-mediated internalization of the conjugate. Linkers can be cleavable or non-cleavable. Cleavable linkers are linkers that are cleaved through intracellular metabolism after internalization, such as cleavage by hydrolysis, reduction, or enzymatic reaction. Non-cleavable linkers are linkers that release the attached payload through lysosomal degradation of the antibody after internalization. Suitable linkers include, but are not limited to, acid-labile linkers, hydrolysis-labile linkers, enzymatically cleavable linkers, reducing labile linkers, self-cleavable linkers, and non-cleavable linkers. Suitable linkers also include, but are not limited to, those that are or contain peptides, glucuronides, succinimide thioesters, polyethylene glycol (PEG) units, hydrazones, mal-caproyl units, dipeptide units, valine-citrulline units and para-aminobenzyl (PAB) units. In some cases, the linker is capable of binding to an antibody or antigen-binding fragment through a lysine residue or a cysteine residue (eg, through cleavage of a disulfide group of the antibody or fragment, or through a cysteine residue incorporated into the antibody or fragment). In some cases, the linker is capable of binding to the antibody or fragment through a glutamine residue, including those obtained by transglutaminase-mediated conjugation.

Exemplary linkers that may be used in the context of this invention include linkers that contain or consist of, for example, MC (6-maleimidocaproyl), MCC (maleimidomethyl cyclohexane-1-carboxylate), MP (maleimidopropanoyl), val-cit (valine -citrulline), val-ala (valine-alanine), ala-phe (alanine-phenylalanine), phe-lys (phenylalanine-lysine), dipeptide cleavage site

- 44 045730 protease-linker, PAB (p-aminobenzyloxycarbonyl), SPP (N-succinimidyl 4-(2pyridylthio)pentanoate), SMCC (N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate), SIAB (N- succinimidyl (4-iodo-acetyl)aminobenzoate) and their variants and combinations.

Additional examples of linkers that can be used in the context of this invention are described, for example, in US Patent No. 7754681 and in Ducry, Bioconjugate Chem., 2010, 21: 5-13, and references cited therein, the contents of which are incorporated herein in their entirety by references in their entirety. In some cases, the linker is or contains a self-cleaving spacer, such as those discussed in Jin, et al., Bioorganic & Medicinal Chemistry, 2012, 20: 34653469, and Wu, et al., Bioorganic & Medicinal Chemistry, 2016, 24: 2697- 2706.

The payload may be associated with an anti-MUC16 antibody or antigen-binding fragment thereof through attachments at specific amino acids within the antibody or antigen-binding molecule. Exemplary amino acid attachments that may be used in the context of this aspect of the invention include, for example, lysine (see, for example, US Pat. No. 5,208,202; US 2010/0129314; Hollander et al., Bioconjugate Chem., 2008), 19: 358 -361; WO 2005/089808; US Patent No. 5714586; US 2013/0101546 and US 2012/0585592), cysteine (see, for example, US 2007/0258987; WO 2013/055993; WO 2013/055990; WO 2013/053873; WO 2013/053872; WO 2011/1 30598; US 2013 /0101546; and US patent No. 7750116), selenocysteine (see, for example, WO 2008/122039; and Hofer et al., Proc. Natl. Acad. Sci., USA, 2008, 105: 12451-12456), formylglycine ( see, for example, Carrico et al., Nat. Chem. Biol., 2007, 3: 321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 110: 46-51, and Rabuka et al., Nat. Protocols, 2012, 10: 1052-1067), unnatural amino acids (see, for example, WO 2013/068874 and WO 2012/166559) and acidic amino acids (see, for example, WO 2012/05982) . Linkers can also be conjugated to antigen binding protein by attachment to carbohydrates (see, for example, US 2008/0305497 and Ryan et al., Food & Agriculture Immunol., 2001, 13: 127-130) and disulfide linkers (see, for example, , WO 2013/085925, WO 2010/010324, WO 2011/018611 and Shaunak et al., Nat. Chem. Biol., 2006, 2: 312-313).

The drug-antibody ratio (DAR) is the average number of drugs conjugated to an antibody or antigen-binding moiety, which has an important impact on the efficacy, activity, and pharmacokinetics of an ADC. In various embodiments of the invention, the DAR is 1, 2, 3, 4, 5, 6, 7, or 8 drug molecules per antibody. In some embodiments, the DAR is from 1 to 4. In certain embodiments, the DAR is from 2 to 4. In some cases, the DAR is from 2 to 3. In some cases, the DAR is from 3 to 4. In some embodiments, of the invention, the DAR is from 1 to 10, from 1 to 20 or from 1 to 30 (that is, from 1 to 30 drug molecules per antibody or antigen-binding fragment).

Therapeutic formula and administration.

This invention relates to pharmaceutical compositions containing the antigen binding molecules of this invention. The pharmaceutical compositions of this invention are formulated with suitable carriers, excipients and other agents that provide improved transport, delivery, tolerability and the like. Many suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™ Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, emulsions such as oil in water or water in oil, emulsions based on carbowax (polyethylene glycols with different molecular weights), semi-solid gels and semi-solid mixtures containing carbowax; see also Powell et al. Compendium of excipients for parenteral formulations PDA (1998) J Pharm Sci Technol 52:238–311.

The dose of antigen binding molecule administered to a patient may vary depending on the age and size of the patient, the target disease, conditions, route of administration, and the like. The preferred dose is usually based on body weight or body surface area. When the bispecific antigen binding molecule of the present invention is used therapeutically in an adult patient, it may be advantageous to administer the bispecific antigen binding molecule of the present invention intravenously, typically in a single dose of about 0.01 to about 20 mg/kg body weight, more preferably from about 0. 02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and duration of treatment may be adjusted. Effective doses and schedules of administration of a bispecific antigen-binding molecule can be determined experimentally; for example, the patient's progress can be monitored through periodic assessment and the dose adjusted accordingly. In addition, interspecies dose scaling can be accomplished using methods well known in the art (eg, Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer the pharmaceutical composition of this invention, for example, encapsulation in liposomes, microparticles, micro

- 45 045730 capsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Routes of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes of administration. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucosal linings (eg, oral mucosa, rectal and intestinal mucosa, and the like), and can be administered together with other biologically active agents. Administration may be systemic or local.

The pharmaceutical composition of this invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, the delivery pen can readily be used to deliver the pharmaceutical composition of the present invention. This syringe pen can be reusable or disposable. A refillable pen typically uses a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition contained in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen can then be reused. The disposable syringe pen does not have a replaceable cartridge. Rather, the disposable pen is filled with a pharmaceutical composition held in a reservoir within the device. Once the reservoir is empty of pharmaceutical composition, the entire device is discarded.

Numerous reusable pens and syringe tubes are useful for subcutaneous delivery of the pharmaceutical composition of this invention. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burgdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG pen ™, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark) , BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name a few. Examples of disposable pens that are used for subcutaneous delivery of the pharmaceutical composition of this invention include, but are not limited to, SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly), syringe SURECLICK™ tube (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA™ pen (Abbott Labs, Abbott Park, IL), to name a few them.

In certain situations, the pharmaceutical composition may be provided in a controlled release system. In one embodiment of the invention, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 201). In another embodiment of the invention, polymeric materials may be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, FL. In yet another embodiment of the invention, the controlled release system can be located close to the target of the composition, thus requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115 -138). Other controlled release systems are discussed in Langer, 1990, Science 249: 1527-1533.

Injectable forms may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions and the like. These injectable forms can be prepared by generally known methods. For example, injectable forms can be prepared, for example, by dissolving, suspending or emulsifying the antibody or salt thereof described above in a sterile aqueous or oily medium commonly used for injection. As the aqueous injection medium, there are, for example, saline solution, isotonic solution containing glucose and other auxiliaries and the like, which can be used in combination with an appropriate solubilizing agent such as alcohol (for example, ethanol), polyalcohol (for example, propylene glycol, polyethylene glycol), nonionic surfactant [for example, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil] and the like. As an oily medium, for example, sesame oil, soybean oil and the like are used which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus obtained is preferably filled into a suitable ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in unit dosage form suitable for the dosage of the active ingredients. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories and the like. The amount of the above-mentioned antibody contained is usually from 5 to 500 mg per dosage form in a unit dose; especially in

- 46 045730 injection form, it is preferable that the above antibody is contained in an amount of from about 5 to about 100 mg and from about 10 to about 250 mg for other dosage forms.

Therapeutic use of antigen-binding molecules.

The present invention includes methods comprising administering to a subject in need thereof a therapeutic composition comprising an anti-MUC16 antibody or antigen binding fragment or bispecific antigen binding molecule that specifically binds to CD3 and MUC16. The therapeutic composition may contain any of the antibodies or bispecific antigen binding molecules as described herein and a pharmaceutically acceptable carrier or diluent. As used herein, the expression subject in need means a person or non-human animal who exhibits one or more symptoms or signs of cancer (for example, a subject expressing a tumor or suffering from any of the types of cancer mentioned herein below) , or who would otherwise benefit from inhibition or reduction of MUC16 activity or depletion of MUC16+ cells (eg, ovarian cancer cells).

The antibodies and bispecific antigen-binding molecules of this invention (and therapeutic compositions containing the same) are useful, in particular, for the treatment of any disease or disorder in which stimulation, activation and/or targeting of the immune response would be beneficial. In particular, the anti-MUC16 antibodies or anti-CD3/anti-MUC16 bispecific antigen binding molecules of the present invention can be used for the treatment, prevention and/or amelioration of any disease or disorder associated with or mediated by MUC16 expression or the activity or proliferation of MUC16+ cells. The mechanism of action by which the therapeutic methods of this invention are achieved involves killing cells expressing MUC16 in the presence of effector cells, for example, through CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms. Cells expressing MUC16 that can be inhibited or killed using the bispecific antigen binding molecules of the present invention include, for example, ovarian cancer cells.

The antigen binding molecules of the present invention can be used to treat a disease or disorder associated with MUC16 expression, including, for example, cancers including ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, mass-forming intrahepatic cholangiocarcinoma, cervical adenocarcinoma, and adenocarcinoma of the gastric tract. In accordance with some embodiments of the present invention, anti-MUC16 antibodies or anti-MUC16/anti-CD3 bispecific antibodies are useful for treating a patient suffering from ovarian cancer. In other related embodiments, the invention provides methods comprising administering an anti-MUC16 antibody or an anti-CD3/anti-MUC16 bispecific antigen binding molecule, as described herein, to a patient suffering from ovarian cancer.

Analytical/diagnostic methods known in the art, such as tumor scanning, etc., can be used to determine whether a patient has an ovarian tumor.

The present invention also provides methods for treating residual cancer in a subject. As used herein, the term residual cancer means the presence or persistence of one or more cancer cells in a subject after treatment with anticancer therapy.

In accordance with some aspects, this invention provides methods for treating a disease or disorder associated with MUC16 expression (eg, ovarian cancer), comprising administering one or more of the anti-MUC16 or bispecific antigen-binding molecules described elsewhere herein to a subject thereafter , the subject has been determined to have prostate cancer. For example, the present invention includes methods of treating ovarian cancer comprising administering an anti-MUC16 antibody or an anti-CD3/anti-MUC16 bispecific antigen binding molecule to a patient after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year or more after the subject received hormonal therapy (eg, androgen deprivation therapy).

Combination therapy and formulations.

This invention relates to methods that involve administering a pharmaceutical composition containing any of the exemplary antibodies and bispecific antigen binding molecules described herein in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents that may be combined or administered in combination with an antigen binding molecule of the present invention include, for example, an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab or panitumumab] or a small molecule EGFR inhibitor [e.g., gefitinib or erlotinib] ), an antagonist of another EGFR family member such as Her2/ErbB2, ErbB3 or ErbB4 (for example, an anti-ErbB2, anti-ErbB3 or anti-ErbB4 antibody or a small molecule inhibitor of ErbB2, ErbB3 or ErbB4 activity), an EGFRvIII antagonist (for example, an antibody that specifically binds EGFRvIII), cMET antagonist (eg anti-cMET antibody), IGF1R antagonist (eg anti-IGFlR

- 47 045730 antibody), B-raf inhibitor (e.g. vemurafenib, sorafenib, GDC-0879, PLX-4720), PDGFR-α inhibitor (e.g. anti-PDGFR-a antibody), PDGFR-β inhibitor (e.g. anti- PDGFR-e antibody), VEGF antagonist (e.g., VEGF decoy, see, e.g., US 7,087,411, (also referred to herein as VEGF inhibitory fusion protein), anti-VEGF antibody (e.g., bevacizumab), small molecule receptor kinase inhibitor VEGF (eg sunitinib, sorafenib or pazopanib)), DLL4 antagonist (eg anti-DLL4 antibody described in US 2009/0142354 such as REGN421), Ang2 antagonist (eg anti-Ang2 antibody described in US 2011/0027286, such as H1H685P), FOLH1 antagonist (PSMA), PRLR antagonist (eg, anti-PRLR antibody), STEAP1 or STEAP2 antagonist (eg, anti-STEAP1 antibody or anti-STEAP2 antibody), TMPRSS2 antagonist (eg, anti-TMPRSS2 antibody) , MSLN antagonist (eg, anti-MSLN antibody), CA9 antagonist (eg, anti-CA9 antibody), uroplakin antagonist (eg, anti-uroplakin antibody), etc. Other agents that can be advantageously administered in combination with antigen-binding molecules according to this of the invention include cytokine inhibitors, including small molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL- 9, IL-11, IL-12, IL-13, IL-17, IL-18 or their respective receptors. The pharmaceutical compositions of this invention (e.g., pharmaceutical compositions containing an anti-CD3/anti-MUC16 bispecific antigen-binding molecule as described herein) can also be administered as part of a therapeutic regimen containing one or more therapeutic combinations selected from ICE: ifosfamide ( eg Ifex®), carboplatin (eg Paraplatin®), etoposide (eg Etopophos®, Toposar®, VePesid®, VP-16); DHAP: dexamethasone (eg, Decadron®), cytarabine (eg, Cytosar-U®, cytosine arabinoside, ara-C), cisplatin (eg, Platinol®-AQ); and ESHAP: etoposide (eg, Etopophos®, Toposar®, VePesid®, VP16), methylprednisolone (eg, Medrol®), high dose cytarabine, cisplatin (eg, Platinol®AQ).

This invention also includes therapeutic combinations containing any of the antigen binding molecules mentioned herein and an inhibitor of one or more of VEGF, Ang2, D114, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-α , PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the above cytokines, wherein the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptide body, nanobody, or antibody fragment ( e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment) or other engineered molecules, e.g. diabodies, triantbodies, tetrabels, minibodies and minimal recognition units). The antigen-binding molecules of this invention can also be administered and/or co-formulated in combination with antivirals, antibiotics, analgesics, corticosteroids and/or NSAIDs. The antigen-binding molecules of this invention can also be administered as part of a treatment regimen that also includes radiation therapy and/or conventional chemotherapy.

Additional therapeutically active component(s) can be administered immediately before, simultaneously with, or shortly after administration of the antigen binding molecule of this invention; (for the purposes of this invention, such administration regimens are considered to be the administration of an antigen-binding molecule in combination with an additional therapeutically active component).

This invention includes pharmaceutical compositions in which the antigen binding molecule of this invention is co-formulated with one or more additional therapeutically active component(s) as described elsewhere herein.

Administration schemes.

In accordance with some embodiments of the present invention, multiple doses of an antigen binding molecule (eg, an anti-MUC16 antibody or a bispecific antigen binding molecule that specifically binds to MUC16 and CD3) may be administered to a subject over a period of time. The methods of this aspect of the invention involve sequentially administering multiple doses of an antigen binding molecule of the invention to a subject. As used herein, sequential administration means that each dose of the antigen binding molecule is administered to a subject at a different point in time, eg, on different days separated by a predetermined interval (eg, hours, days, weeks, or months). The present invention includes methods that include sequentially administering to a patient a single initial dose of an antigen binding molecule, followed by one or more secondary doses of the antigen binding molecule, and optionally followed by one or more tertiary doses of the antigen binding molecule.

The terms initial dose, secondary doses and tertiary doses refer to the time sequence of administration of the antigen binding molecule of this invention. Thus, the starting dose is the dose that is administered at the beginning of the course of treatment (also called the initial dose); secondary doses are doses administered after the initial dose; and tertiary doses are doses administered after secondary doses. Initial, secondary and tertiary doses may contain the same amount of antigen binding molecule, but usually may

- 48 045730 differ from each other in terms of frequency of administration. However, in certain embodiments of the present invention, the amount of antigen-binding molecule contained in the initial, secondary and/or tertiary doses differs from each other (eg, adjusted up or down as necessary) over the course of treatment. In some embodiments, two or more (eg, 2, 3, 4, or 5) doses are administered early in the course of treatment as loading doses, followed by doses that are administered less frequently (eg, maintenance doses).

In one illustrative embodiment of the present invention, each second and/or third dose is administered every 1 to 26 weeks (eg, 1, 11/2, ₂ , 21/2, 3, 31/2 _, 4, 41/2, 5, 51 /2, 6, 61/ ₂ , 7, 71/2, 8, 81/2, 9, 91/2, 10, 101/2, 11, 111/2, 12, 121/2, 13, 131/2 , 14, 141/2, 15, 151/2, 16, 161/2, 17, 171/2, 18, 181/2, 19, 191/2, 20, 201/2, 21, 211/2, 22 , 221/2, 23, 231/2, 24, 241/2, 25, 251/2, 26, 261/2 and more) after the immediately preceding dose. The phrase immediately preceding dose, as used herein, means, in a multiple administration sequence, the dose of an antigen binding molecule that is administered to a patient before the next dose in the sequence without intervening doses.

Methods according to this aspect of the invention may include administering to the patient any number of secondary and/or tertiary doses of an antigen binding molecule (eg, an anti-MiC16 antibody or a bispecific antigen binding molecule that specifically binds to MUC16 and CD3). For example, in certain embodiments of the invention, only one secondary dose is administered to the patient. In other embodiments, the patient is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses. Likewise, in certain embodiments of the invention, only one tertiary dose is administered to the patient. In other embodiments, the patient is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses.

In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks after the immediately preceding dose. Likewise, in embodiments of the invention that include multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which secondary and/or tertiary doses are administered to the patient may vary during the course of treatment. The frequency of administration may also be adjusted during the course of treatment by the attending physician depending on the needs of the individual patient after clinical examination.

Diagnostic use of antibodies.

The anti-MiC16 antibodies of the present invention can also be used to detect and/or measure MUC16 or MiC16-expressing cells in a sample, such as a biological sample, for diagnostic purposes. For example, an anti-MiC16 antibody or fragment thereof can be used to diagnose a condition or disease characterized by aberrant expression (eg, overexpression, underexpression, lack of expression, etc.) of MUC16. Exemplary diagnostic assays for MUC16 may include, for example, contacting a sample obtained from a patient with an anti-MiC16 antibody of the present invention, wherein the anti-MiC16 antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-MiC16 antibody may be used in diagnostic applications in combination with a secondary antibody that is itself labeled for detection. The detectable label or reporter molecule may be a radioisotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S, or ¹²⁵ I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase or luciferase. Another exemplary diagnostic use of the anti-MiC16 antibodies of the present invention includes ⁸⁹ Zr-labeled, such as ⁸⁹ Zr-desferrioxamine-labeled, antibody for the purpose of non-invasively identifying and tracking tumor cells in a subject [eg, positron emission tomography (PET) imaging ( see, for example, Tavare, R. et al. Cancer Res 2016 Jan 1; 76 (1): 73-82, and Azad, V. V. et al. Oncotarget. 2016 Mar 15; 7 (11): 12344-58). Specific typical assays that can be used to detect or measure MUC16 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in MUC16 diagnostic assays in accordance with this invention include any tissue or fluid sample that can be obtained from a patient that contains detectable amounts of MUC16 protein or fragments thereof under normal or pathological conditions. Typically, MUC16 levels in a particular sample obtained from a healthy patient (eg, a patient not suffering from a disease or condition associated with abnormal MUC16 levels or activity) will be measured for the purpose of initially establishing a baseline or standard MUC16 level. This baseline MUC16 level can then be compared to MUC16 levels measured in samples obtained from individuals suspected of having a MUC16-related disease (eg, a tumor containing MUC16-expressing cells) or condition.

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Examples of tissue or fluid samples include, but are not limited to, plasma, serum, ascites, ovary, uterus, cervix, liver, bladder, pancreas, stomach, small or large intestine, gall bladder, breast, lung, kidney, salivary glands, and lacrimal glands or any malignant epithelioid formation thereof. Additional examples of tissue or fluid samples include, but are not limited to, papillary serous cervical cancer, endometrial adenocarcinoma, clear cell bladder adenocarcinoma, seminal vesicle carcinoma, gastric carcinoma, colorectal adenocarcinoma, and epithelioid mesothelioma. It is contemplated that any fluid or tissue sample that contains detectable amounts of MUC16 protein or fragments thereof may be subjected to the detection methods described herein. The described methods can be used to monitor the development and progression of malignant diseases or to distinguish between normal and disease states. As such, the described methods can be used to detect or monitor cancers such as ovarian cancer, bladder cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, mass-forming intrahepatic cholangiocarcinoma, cervical adenocarcinoma, and gastric tract adenocarcinoma.

Examples

The following examples are provided to provide those skilled in the art with complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of the invention to what the inventors believe to be their invention. Efforts have been made to ensure accuracy in terms of numbers used (e.g. amounts, temperatures, etc.), but some experimental errors and deviations must be taken into account. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

Example 1. Preparation of anti-MUC16 antibodies.

Ahtu-MUC16 antibodies are produced by immunizing a genetically modified mouse with the human MUC16 antigen or immunizing an engineered mouse containing DNA encoding the human immunoglobulin heavy and kappa light chain variable regions with the human MUC16 antigen.

Genetically modified mice were immunized with hMUC16.nub (a truncated format covering the last five SEA domains of mucin-1b (SEQ ID: 1902)) or immunized with an hMUC16-expressing cell line such as OVCAR-3 cells. SEQ ID NO: 1902 contains residues 13810-14451 of SEQ ID NO: 1899, as well as C-terminal tags. Following immunization, splenocytes were collected from each mouse and (1) fused with mouse myeloma cells to maintain their viability and form hybridoma cells and screened for MUC16 specificity, or (2) cell sorted (as described in US 2007/0280945A1) using a fragment of human MUC16 as a sorting reagent that binds and identifies reactive antibodies (antigen-positive B cells).

Chimeric antibodies to MUC16 were originally isolated and had a human variable region and a mouse constant region. Antibodies have been characterized and selected for desired characteristics including affinity, selectivity, etc. When necessary, mouse constant regions were replaced with the desired human constant region, eg, wild-type or modified IgG1 or IgG4 constant region, to generate a fully human anti-MUC16 antibody. Although the selected constant region may vary depending on the specific application, the variable region provides high affinity antigen binding and target specific characteristics. Antibody name designations such as H1H8755P and H1M7129N designate fully human HH antibodies or chimeric human variable/mouse constant region H1M antibodies. Antibodies identified by the hybridoma method are designated by antibody identification numbers ending in N or N2. Antibodies identified by the B cell sorting method are designated by antibody identification numbers ending in P or P2.

Certain biological properties of exemplary anti-MUC16 antibodies produced according to the methods of this example are described in detail in the examples below.

Amino acid and nucleic acid sequences of the variable region of the heavy and light chains of anti-MUC16 antibodies.

In table 1 shows the amino acid sequence identifiers of the variable regions and CDRs of the heavy and light chains of selected anti-MUC16 antibodies according to this invention. The corresponding nucleic acid sequence identifiers are given in Table. 2.

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Table 1

Amino acid sequence identifiers

SEQ ID NO: Antibody designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 Н1Н8755Р 2 4 6 8 10 12 14 16 Н1Н8767Р 18 20 22 24 26 28 thirty 32 Н1Н8770Р 34 36 38 40 42 44 46 48 Н1Н8783Р 50 52 54 56 58 60 62 64 Н1Н8790Р 66 68 70 72 74 76 78 80 Н1Н8794Р 82 84 86 88 90 92 94 96 Н1Н8794Р2 82 84 86 88 858 860 862 864 Н1Н8799Р 98 100 102 104 106 108 110 112 Н1Н8799Р2 98 100 102 104 170 172 174 176 Н1Н8804Р 114 116 118 120 122 124 126 128 Н1Н8808Р 130 132 134 136 138 140 142 144 Н1Н8810Р 146 148 150 152 154 156 158 160 Н1Н8813Р 162 164 166 168 170 172 174 176 H1M7129N 178 180 182 184 186 188 190 192 H1M7137N 194 196 198 200 394 396 398 400 H1M9519N 202 204 206 208 210 212 214 216 H1M9521N 218 220 222 224 226 228 230 232 H1M9528N 234 236 238 240 242 244 246 248 H2M7128N 250 252 254 256 1936 1938 1940 1942 H1M7130N 1944 1946 1948 1950 1952 1954 1956 1958 H2M7131N 258 260 262 264 266 268 270 272 H2M7133N 274 276 278 280 1936 1938 1940 1942 H2M7134N 282 284 286 288 290 292 294 296 H2M7135N 298 300 302 304 306 308 310 312 H2M7138N 314 316 318 320 322 324 326 328 H2M9538N 330 332 334 336 338 340 342 344 H3M9524N 346 348 350 352 354 356 358 360 H3M9525N 362 364 366 368 370 372 374 376 H3M9529N 378 380 382 384 386 388 390 392

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table 2

Nucleic acid sequence identifiers

SEQ ID NO: Antibody designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 Н1Н8755Р 1 3 5 7 9 eleven 13 15 Н1Н8767Р 17 19 21 23 25 27 29 31 Н1Н8770Р 33 35 37 39 41 43 45 47 Н1Н8783Р 49 51 53 55 57 59 61 63 Н1Н8790Р 65 67 69 71 73 75 77 79 Н1Н8794Р 81 83 85 87 89 91 93 95 Н1Н8794Р2 81 83 85 87 857 859 861 863 Н1Н8799Р 97 99 101 103 105 107 109 111 Н1Н8799Р2 97 99 101 103 169 171 173 175 Н1Н8804Р 113 115 117 119 121 123 125 127 Н1Н8808Р 129 131 133 135 137 139 141 143 Н1Н8810Р 145 147 149 151 153 155 157 159 Н1Н8813Р 161 163 165 167 169 171 173 175 H1M7129N 177 179 181 183 185 187 189 191 H1M7137N 193 195 197 199 393 395 397 399 H1M9519N 201 203 205 207 209 211 213 215 H1M9521N 217 219 221 223 225 227 229 231 H1M9528N 233 235 237 239 241 243 245 247 H2M7128N 249 251 253 255 1935 1937 1939 1941 H1M7130N 1943 1945 1947 1949 1951 1953 1955 1957 H2M7131N 257 259 261 263 265 267 269 271 H2M7133N 273 275 277 279 1935 1937 1939 1941 H2M7134N 281 283 285 287 289 291 293 295 H2M7135N 297 299 301 303 305 307 309 311 H2M7138N 313 315 317 319 321 323 325 327 H2M9538N 329 331 333 335 337 339 341 343 H3M9524N 345 347 349 351 353 355 357 359 H3M9525N 361 363 365 367 369 371 373 375 H3M9529N 377 379 381 383 385 387 389 391

Example 2: Ahtu-MUC16 antibodies specifically bind to endogenously expressed hMUC16 on the OVCAR-3 cell line.

The ability of anti-MUC16 antibodies to bind specifically to endogenously expressed MUC16 on a human ovarian carcinoma cell line (OVCAR-3) was assessed using an electrochemiluminescence-based detection assay (Meso Scale of Detection (MSD), Rockville, MD).

Briefly, OVCAR-3 and a control ovarian adenocarcinoma cell line, SK-OV-3, which has no detectable expression of hMUC16, were washed in 1xPBS supplemented with Ca ²⁺ /Mg ²⁺ (Irvine Scientific, Santa Ana, CA) and then incubated in cell dissociation buffer without enzymes (Millipore, Billerica, MA) for 10 minutes at 37°C. Detached cells were then washed once in 1xPBS supplemented with Ca ²⁺ /Mg ²⁺ and counted (Cellometer Auto T4 cell counter, Nexcelom Bioscience, Lawrence, MA). Approximately 1.0 x 104 cells were seeded into MULTI-ARRAY 96-well carbon electrode (MSD) plates and incubated for 1 hour at 37°C. Nonspecific binding sites were then blocked with 2% BSA (w/v) in PBS for 1 hour.

- 52 045730 at room temperature. Serial dilutions of anti-MiC16 or control antibodies (0.85 pM - 50 nM) and buffer controls without antibodies were then added to the cells bound to the plates and incubated for 1 hour at room temperature (RT). The plates were then washed to remove unbound antibodies using an AquaMax2000 immunoplate washer (MDS Analytical Technologies, Sunnyville, CA). Plate-bound antibodies were detected using SULFO-TAG™ anti-human kappa light chain-conjugated antibody (Regeneron) or SULFO-TAG™ anti-mouse IgG-conjugated antibody (Jackson Immunoresearch, West Grove, PA). within 1 hour with CT scan. After washing, the plates were developed using reading buffer (MSD) according to the manufacturer's protocol, and luminescent signals were recorded using a SECTOR Imager 6000 (MSD).

Luminescence intensity, measured in relative light units (RLU), for the two cell lines was recorded to indicate the binding intensity of each antibody. The ratio of signal detected upon binding of 1.9 nM or 16.7 nM anti-MUC16 antibody to OVCAR-3 compared to SK-OV-3 was reported as an indicator of binding specificity and efficiency (Table 3).

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Table 3

Ratio of cell binding of anti-Myc16 antibodies to endogenous expression of hMUC16 OVCAR-3 compared to hMUC16-negative SK-OV-3 cell lines

Ratio: anti-miC16 binding OVCAR-3/SK-OV3

Antibody ID [At]: 1.9 nM [At]: 16.7 nM Anti-Mis16 antibody hybridoma (HIM, H2M, NZM) H2M7128N 59 31 H1M7129N 68 19 H2M7131N 86 37 H2M7133N 69 33 H2M7134N 68 29 H2M7135N thirty 5 H1M7137N 77 29 H2M7138N 82 50 H3M7132N 89 38 H1M9519N 109 78 H1M9521N 132 107 H3M9524N 81 57 H3M9525N 137 51 H1M9528N 153 120 H3M9529N 143 99 H2M9538N 89 26 Human Fc anti-miC16 antibodies (H1H) Н1Н8755Р 13 5 Н1Н8767Р 4 N.S. Н1Н8770Р 9 3 Н1Н8783Р eleven 4 Н1Н8790Р 8 3 Н1Н8794Р 8 3 Н1Н8794Р2 5 N.S. Н1Н8799Р 10 4 Н1Н8799Р2 10 4 Н1Н8804Р 8 3 Н1Н8808Р 4 N.S. Н1Н8810Р 8 3 Н1Н8813Р eleven 5

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H1H9519N 41 thirty H1H9521N thirty 28 H1H9524N 20 48 H1H9525N 4 21 H1H9528N 41 18 H1H9529N 109 74 H1H9538N 12 13 Controls Isotype control antibody mlgGl N.S. N.S. Isotype control antibody m!gG2a N.S. N.S. hlgGl isotype control antibody N.S. N.S.

NS=non-specific - ratio at 1.9 nM or 16.7 nM <2.5 fold.

Isotypes: H1H: hIgG1; HIM: mIgG1; H2M: mIgG2; H3M: mIgG3.

Note: Variation in binding intensity ratio between the same antibody expressed with mouse Fc and human Fc is due to the use of different SULFO-TAG secondary detection reagents.

As can be seen from the results in table. 3, the majority of the anti-MUC16 antibodies of the present invention bind specifically to OVCAR-3 at both high (16.7 nM) and low (1.9 nM) antibody concentrations. Isotype control antibodies mIgG1, mIgG2a and hIgG1 did not show specific binding to either the OVCAR-3 or SK-OV-3 cell line. In addition, there is evidence that the method is sensitive, as some antibodies that did not show binding to soluble monomeric human MUC16 protein in a surface plasmon resonance binding assay (see Example 4 below) showed specific binding to endogenous human MUC16 expressed on OVCAR-3 cells in this cell-based binding assay.

Example 3. Preparation of bispecific antibodies that bind to cell-specific (MUC16) and CD3 of the ovary.

The present invention provides bispecific antigen binding molecules that bind to CD3 and MUC16; such bispecific antigen binding molecules are also referred to herein as bispecific anti-MUC16/anti-CD3 or anti-MUC16xCD3 molecules. The anti-MUC16 portion of the anti-MUC16/anti-CD3 bispecific molecule is useful for targeting tumor cells that express MUC16 (also known as CA-125), and the anti-CD3 portion of the bispecific molecule is useful for activating T cells. Simultaneous binding to MUC16 on the tumor cell and CD3 on the T cell facilitates targeted killing (cell lysis) of the target tumor cell by the activated T cell.

Bispecific antibodies containing an anti-MUC16-specific binding domain and an anti-CD3-specific binding domain were constructed using standard techniques, with the anti-MUC16 antigen-binding domain and the anti-CD3 antigen-binding domain each containing different, distinct HCVRs paired with a common LCVR . In the exemplary bispecific antibodies, molecules were constructed using a heavy chain from an anti-CD3 antibody, a heavy chain from an anti-MUC16 antibody, and a common light chain from an anti-MUC16 antibody. In other cases, bispecific antibodies can be constructed using a heavy chain from an anti-CD3 antibody, a heavy chain from an anti-MUC16 antibody, and a light chain from an anti-CD3 antibody, or an antibody light chain that is known to be heterogeneous or effectively paired with different arms of the heavy chain.

The resulting bispecific antibodies described in the following examples consist of anti-CD3 binding arms having different binding affinities for human soluble heterodimeric hCD3 ε/δ protein (as described in Example 15 herein); and human MUC16 (see examples 1-2 above). The exemplified bispecific antibodies were made having an IgG1 Fc domain (BSMUC16/CD3-001, -002, 003, and -004) or a modified (chimeric) domain

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Fc IgG4 (BSMUC16/CD3-005) as set forth in US Patent Application Publication No. US20140243504A1 published August 28, 2014.

A brief description of the components of the antigen-binding domains of various anti-MUC16xCD3 bispecific antibodies constructed is presented in table. 4.

Table 4

Summary of component parts of selected anti-MUC16xCD3 bispecific antibodies

Bispecific Antibody ID Anti-MiS16 Ahth-CD3 Common Light chain variable region Antigen binding domain Antigen binding domain Heavy chain variable region Heavy chain variable region BSMUC16/CD3-001 Н1Н8767Р (SEQUIDNO: 18) CD3-VH-G (SEQIDNO: 1730) Н1Н8767Р (SEQ Ш NO: 26) BSMUC16/CD3-002 Н1Н8767Р (SEQUIDNO: 18) CD3-VH-G5 (SEQIDNO: 1762) Н1Н8767Р (SEQ Ш NO: 26) BSMUC16/CD3-003 Н1Н8767Р (SEQUIDNO: 18) CD3-VH-G9 (SEQIDNO: 1778) Н1Н8767Р (SEQ Ш NO: 26) BSMUC16/CD3-004 Н1Н8767Р (SEQUIDNO: 18) CD3-VH-G10 (SEQIDNO: 1786) Н1Н8767Р (SEQ Ш NO: 26) BSMUC16/CD3-005 Н1Н8767Р (SEQUIDNO: 18) CD3-VH-G20 (SEQIDNO: 1866) Н1Н8767Р (SEQ Ш NO: 26)

Light chains listed in table. 4 were common to both the CD3 and MUC16 targeting arms of bispecific antibodies. In table 1 and 2 show the amino acid and nucleic acid sequence identifiers, respectively, for the various heavy chain variable regions and their corresponding CDR arms of the anti-MUC16 bispecific antibodies of this example. In table 23 and 23 show the amino acid and nucleic acid sequence identifiers, respectively, for the various heavy chain variable regions and their corresponding CDR arms of the anti-CD3 bispecific antibodies of this example.

Example 4. Binding affinity and kinetic constants of surface plasmon resonance origin based on human monoclonal anti-MUC16 monospecific and anti-MUC16xCD3 bispecific antibodies.

Binding affinities and kinetic constants of human anti-MUC16 antibodies were determined using real-time surface plasmon resonance (SPR; Biacore 4000 or Biacore T-200, GE Healthcare Life Sciences, Pittsburgh, PA) at 25°C. The Ahtu-MUC16 antibodies tested in this example were divalent monospecific binders to MUC16 (expressed with the hIgG1 (H1H), mIgG1 (HIM), mIgG2 (H2M) or mIgG3 (H3M) constant region) or bispecific antibodies consisting of anti -MUC16 binding domain and anti-CD binding domain. Antibodies were captured on the surface of a CM4 or CM5 Biacore sensor (GE Healthcare Life Sciences) derivatized by amine coupling with an anti-human Fc monoclonal antibody (GE, #BR-1008-39) or an anti-mouse Fc monoclonal antibody (GE, #BR-1008-39). 38). Various concentrations of soluble monomeric human MUC16 in a truncated format covering the last five domains of Mucin-16 SEA (hMUC16.mmh or MUC16 nub, SEQ ID: 1902) were injected over an anti-MUC16 antibody capture surface at a flow rate of 50 μl/min (Biacore T -200) or 30 µl/min (Biacore 4000). Antibody-reagent association was monitored for 4 minutes, and dissociation was monitored for 6-10 minutes. All binding studies were performed in HBS-ET buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.05% v/v P20 surfactant).

Kinetic rate constants for association (ka) and dissociation (kd) were determined by fitting real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software. Binding dissociation equilibrium constants (KD) and dissociative half-lives (1%) were calculated from the kinetic rate constants as:

kd Zn(2)

K _D (Μ) = — , and t4 (min) = ——— ka 60 * kd

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The kinetic parameters of binding for monospecific anti-MiC16 antibodies with a monomeric fragment of the human MUC16 protein are shown below in table. 5A and 5B. The kinetic parameters of binding of anti-MUC16/anti-CD3 bispecific antibodies to monomeric human MUC16 protein are shown in table below. 6.

Table 5A

Binding affinity of Biacore hybridoma anti-MUC16 antibodies (H1M, H2M and H3M) to hMUC16 fragment at 25°C

Antibody ID ka (1/Ms) kd (1/s) K _D (M) t 1/2 (min) H2M7128N 1.89E+05 6.40E-04 3.39E-09 18 H1M7129N 5.31E+04 1.04E-04 1.97E-09 111 H2M7131N 6.47E+04 1.62E-04 2.51E-09 71 H3M7132N 2.57E+04 1.96E-04 7.62E-09 59 H2M7133N 1.67E+05 3.77E-04 2.26E-09 31 H2M7134N 6.55E+04 1.62E-04 2.47E-09 71 H2M7135N 5.10E+04 2.18E-04 4.27E-09 53 H1M7137N 5.30E+04 9.09E-05 1.72E-09 127 H2M7138N 7.41E+04 9.25E-05 1.25E-09 125 H1M9519N OS OS OS OS H1M9521N OS OS OS OS H3M9524N OS OS OS OS H3M9525N OS OS OS OS H1M9528N OS OS OS OS H3M9529N OS OS OS OS

OS: no binding.

Table 5B

Binding affinity of human Biacore Fc anti-MUC16 antibodies (H1H) to hMUC16 fragment at 25°C

Antibody ID ka (1/Ms) kd (1/s) K _D (M) ti/2 (min) Н1Н8755Р 5.22E+05 1.49E-04 2.86E-10 77 Н1Н8767Р 1.17E+05 4.18E-04 3.58E-09 28 Н1Н8770Р 2.47E+05 3.08E-04 1.25E-09 38 Н1Н8783Р 1.74E+05 1.07E-04 6.14E-10 108 Н1Н8790Р 1.01E+05 7.61E-04 7.53E-09 15 Н1Н8794Р 3.62E+05 2.79E-04 7.71E-10 41 Н1Н8799Р 7.90E+04 3.66E-04 4.63E-09 32 Н1Н8799Р2 7.58E+04 3.73E-04 4.92E-09 31 Н1Н8804Р 4.94E+04 6.07E-04 1.23E-08 19 Н1Н8808Р 4.12E+03 2.16E-04 5.24E-08 54 Н1Н8810Р 5.77E+04 3.16E-04 5.48E-09 37 Н1Н8813Р 5.32E+04 2.32E-04 4.35E-09 50

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Table 6

Binding affinity of Biacore anti-MUC16/anti-CD3 bispecific antibodies to hMUC16 fragment at 25°C

Bispecific Antibody ID ka (1/Mc) kd(1/c) _KD (M) t 1/2 (min) BSMUC16/CD3-001 9.48E+04 5.86E-04 6.18E-09 20 BSMUC16/CD3-005 9.41E+04 5.64E-04 6.00E-09 21

As the results show, most of the anti-MUC16 antibodies of this invention bind to soluble human MUC16 protein, some displaying sub-nanomolar affinity. Several antibodies (H1M9519N, H1M9521N, H3M9524N, H3M9525N, H1M9528N, H3M9529N) did not show binding to a truncated format spanning the last five SEA domains by surface plasmon resonance, but did show specific binding to endogenous human MUC16 expressed on OVCAR-3 cells based on cell binding assay. The Ahtu-MUC16xCD3 bispecific antibodies of the present invention also bound to the soluble truncated human MUC16 protein exhibiting nanomolar affinity in this assay.

Example 5: Additional Binding, T Cell Activation, and Cytotoxicity Properties of Exemplary Bispecific Antibodies.

In this example, the ability of MUC16xCD3 bispecific antibodies to bind to CD3-expressing (human T cell) human cell lines, compared to binding to a target-specific (MUC16-specific) cell line, was determined by FACS. In addition, the ability of these bispecific antibodies to activate target-specific cell lines (MUC16-specific) was also compared in a similar assay.

Titration of binding of exemplary bispecific antibodies measured by FACS assay A. Briefly, flow cytometry analysis (i.e., fluorescence-activated cell sorting, or FACS) was used to determine the binding of bispecific antibodies to Jurkat cells or cells expressing human MUC16, followed by detection using phycoerythrin (PE)-labeled or APC-labeled anti-human IgG antibody. Briefly, 2 x 10 ⁵ cells/well were incubated for 30 minutes at 4°C with serial dilution ranging from 1.33E-07M to 8.03E-12M of each test antibody or isotype control (antibody of the same isotype that binds another antigen to no cross-reactivity to MUC16 or CD3). After incubation, cells were washed twice with cold PBS containing 1% filtered FBS, and PE-conjugated or APC-conjugated anti-human secondary antibody was added to the cells and incubated for an additional 30 minutes. Wells containing no antibodies or only secondary antibodies were also used as controls. After incubation, cells were washed, resuspended in 200 μl of cold PBS containing 1% filtered FBS, and analyzed by flow cytometry on a BD FACS Canto II; see table 7A.

B. In separate experiments with conditions similar to those described above, flow cytometry (or FACS) analysis was used to determine the binding of selected bispecific antibodies to Jurkat, PEO-1, OVCAR3-Luc and cynomolgus T cells. For titration assays, serial dilution of selected MUC16xCD3 bispecific antibodies, a first isotype control antibody (a human chimeric IgG4 antibody that binds an irrelevant human antigen without cross-reactivity with human or cynomolgus CD3), and a second isotype control antibody (a human chimeric IgG4 antibody that binds an irrelevant human antigen without cross-reactivity with human MUC16), ranging from 66.6 nM to 0.001 nM; see table 7B and FIG. 11A-11C.

For FACS analysis, cells were gated along the forward scatter height versus the forward scatter region to select individual events, and then laterally and forward scatter. EC50 for cell binding titration was determined using PRISM™ software (GraphPad Software, Inc., La Jolla, CA). Values were calculated using 4-parameter nonlinear regression analysis. (Liu, J., et al. 2005, Biotechnol Letters 27: 18211827). The EC50 value represents the concentration of the test antibody at which 50% of its maximum binding is observed.

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Table 7A

FACS binding on CD3 and MiC16-specific cell lines

Bispecific Antibody ID Anti-CD3 binding arm FACS binding titration EC ₅₀ [M] [01] [01] Jurkat OVCAR3 (MUC16+) BSMUC16/CD3-001 CD3-VH-G 3.21E-09 1.20E-09 BSMUC16/CD3-002 CD3-VH-G5 Very weak 2.69E-09

Table 7B

FACS binding on CD3 and MUC16-specific cell lines

Bispecific Antibody ID Ahth-CD3 binding arm FACS binding titration ec ₅₀ [M] REO-1 (MUC16+) OVCAR3-Luc (MUC16+) Jurkat Cynomolgus T cells BSMUC16/CD3-001 CD3-VH-G 3.02E-09 1.32E-09 6.44E-09 1.56E-08 BSMUC16/CD3-002 CD3-VH-G5 3.13E-09 Not tested 3.01E-07 No binding BSMUC16/CD3- 005 CD3-VH-G20 2.63E-09 1.47E-09 6.26E-08 1.17E-06

As shown in table. 7A and 7B, the CD3 binding arms of each MUC16 bispecific antibody depict the range of cell binding to human and monkey CD3-expressing T cells (eg, 3.2 nM EC50 to very weak binding). The BSMUC16/CD3-001 bispecific antibody showed high binding to CD3-expressing cells (ie, <7 nM), while the BSMUC16/CD3-002 bispecific antibody showed weak or no binding to human and monkey CD3-expressing cells. Immeasurable binding or lack of measurable binding in a FACS assay or equivalent assay refers to a binding interaction between an antibody and its target antigen that is outside the detection limit of the assay (eg, at or above 1 μM). The bispecific antibodies tested showed similar cell binding on cell lines expressing MUC16, confirming that bispecific pairing with individual CD3 arms exhibiting high or weak (or unmeasurable) interactions with CD3 did not affect or reduce target-specific binding (MUC16- specific binding was less than or equal to 3 nM (high binding) in these examples.The first control antibody did not bind to CD3+ cells and the second control antibody did not bind to MUC16+ cells; see also Fig. 11A-11C.

Antibodies exhibiting little or no detectable binding to human CD3 are still considered useful for avidity-driven bispecific pairing and have been further tested for cytotoxicity in vitro (see below) and in vivo (Example 8).

T cell activation and tumor-specific cytotoxicity exhibited by bispecific antibodies when measured in vitro A. Specific killing of MUC16-expressing tumor target cells in the presence of CD3 based on bispecific antibodies was monitored by flow cytometry. As previously reported, bispecific antibodies exhibited differential binding to CD3 protein and CD3-expressing cell lines (ie, very weak or strong binding). These same bispecific antibodies were tested for their ability to induce naïve human T cells to redirect killing to target expressing cells.

Briefly, MUC16-expressing (OVCAR3) cell lines were labeled with 1 μM fluorescent tracking dye Violet Cell Tracker. After labeling, cells were plated overnight at 37°C. Separately, human PBMCs were seeded in RPMI medium supplemented with 1 x ¹⁰⁶ cells/ml and incubated overnight at 37°C to enrich lymphocytes by depleting adherent macrophages, dendritic cells, and some monocytes. The next day, target cells were co-incubated with attached cell-depleted unstimulated PBMCs (effector/target cell ratio 4:1) and serial dilutions of appropriate bispecific antibodies or isoti

- 59 045730 peak control (concentration range: bb.7 nM to 0.25 pM) for 4 8 hours at 37°C. Cells were recovered from cell culture plates using enzyme-free cell dissociation buffer and analyzed by FACS. See the results presented in table. 8A.

B. In similar studies, MUC16-expressing (PEO-1 or OVCAR3-Luc) cell lines were labeled, plated, and incubated overnight as described. Serial dilutions of MUC16xCD3 bispecific antibodies or isotype control were co-incubated; see the results shown in table. 8B and 8C and FIG. 12A-12B.

For FACS analysis, cells were stained dead/live with far red cell tracker (Invitrogen). 5x105 counting beads were added to each well immediately prior to FACS analysis. 1x104 beads were collected for each sample. To assess specificity of killing, cells were gated on living populations labeled in purple. The percentage of the population alive was recorded and used to calculate normalized survival.

T cell activation was assessed by incubating cells with directly conjugated antibodies to CD2, CD69 and/or CD25, and reporting the percentage of early activated (CD69+) T cells and/or late activated (CD25+) T cells out of total T cells (CD2+).

As the results in table show. 8A-8C, depletion of MUC16-expressing cells was observed with bispecific anti-MUC16xCD3. All bispecific antibodies tested activated and directed human T cells to deplete target cells with EC ₅₀ in the picomolar range. In addition, the observed target cell lysis (depletion) was associated with upregulation of CD69 (or CD25) on CD2+ T cells, also with picomolar (pM) EC ₅₀ .

Importantly, the results of this example also show that a bispecific antibody constructed with CD3 binding arms that exhibited weak or no measurable binding to CD3 protein or CD3-expressing cells (i.e., CD3-VH-G5) still retained ability to activate T cells and exhibited potent cytotoxicity of tumor antigen-expressing cells.

Table 8A

Cytotoxicity and T cell activation properties of selected MUC16xCD3 bispecific antibodies

Bispecific Antibody ID Ahth-CD3 binding arm OVCAR3 cell depletion EC ₅₀ [M] T cell activation (CD69 up-regulation) EC ₅₀ [M] BSMUC16/CD3-001 CD3-VH-G 2.24E-11 5.88E-12 BSMUC16/CD3-002 CD3-VH-G5 3.06E-11 1.01E-11

Table 8B

Cytotoxicity and T cell activation properties of selected MUC16xCD3 bispecific antibodies

Bispecific Antibody ID PEO-1 cell depletion EC ₅₀ [M] T cell activation (CD69 up-regulation) EC50 [M] T cell activation (CD25 up-regulation) EC50 [M] BSMUC16/CD3-001 2.56E-11 8.34E-12 3.90E-11 BSMUC16/CD3-002 6.75E-11 1.34E-11 8.89E-11 BSMUC16/CD3-005 7.74E-11 1.72E-11 1.06E-10

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Table 8C

Cytotoxicity and T cell activation properties of selected MUC16xCD3 bispecific antibodies

Bispecific Antibody ID OVCAR3-Luc cell depletion EC ₅₀ [M] T cell activation (CD69 up-regulation) EC ₅₀ [M] T cell activation (CD25 up-regulation) EC ₅₀ [M] BSMUC16/CD3-001 1.54E-11 2.98E-12 3.06E-11 BSMUC16/CD3-005 5.16E-11 1.54E-11 1.17E-10

Example 6. Epitope mapping based on hydrogen/deuterium (H/D) exchange of anti-Muc16 antibodies H4sH8767P, H1H8794P2 and H1H8799P2 binding to part of the C-terminal domain of hMUC16.

Experiments were conducted to determine the amino acid residues of MUC16 within the five C-terminal SEA domains (SEQ ID NO: 1902, hereafter referred to as hMUC16.nub) with which the anti-MUC16 antibodies H4sH8767P, H1H8794P2 and H1H8799P2 interact. Hydrogen/deuterium (H/D) epitope mapping with mass spectrometry (HDX-MS) was used for this purpose. A general description of the H/D exchange method is given in Ehring (1999) Analytical Biochemistry 267 (2):252-259; and Engen and Smith (2001) Anal. Chem. 73: 256A-265A.

HDX-MS experiments were performed on a single Waters HDX/MS platform consisting of a Leaptec HDX PAL system for deuterium labeling, a Waters Acquity M-Class (auxiliary solvent manager) for digestion and sample loading, a Waters Acquity M-Class (μBinary solvent manager) for the analytical column gradient and a Synapt G2-Si mass spectrometer for measuring the peptic mass of the peptide.

The labeling solution was prepared in 10 mM PBS buffer in D ₂ O at pD 7.0. For deuterium labeling, 3.8 μl of hMUC16.nub (12 pmol/μl) or hMUC16.nub premixed with anti-MUC16 antibody H4sH8767P, H1H8794P2, or H1H8799P2 in a 2:1 molar ratio was incubated with 56.2 μl. D ₂ O labeling solution at various times (eg: non-deuterated control=0 s; deuterium labeling: 1 min and 20 min). Deuteration was quenched by transferring 50 μl of sample to 50 μl of pre-chilled quenching buffer (0.2 M TCEP, 6 M guanidine chloride in 100 mM phosphate buffer, pH 2.5) and the mixed sample incubated at 1.0°C for two minutes. The quenched sample was then introduced into Waters HDX Manager for online pepsin/protease XIII digestion. Digested peptides were captured on an ACQUITY UPLC VEN C18 1.7 µm VanGuard 2.1 x 5 mm precolumn at 0°C and eluted onto an ACQUITY UPLC VEN C18 analytical column (1.7 µm, 1.0 x 50 mm) for a 9 min gradient separation 5%-40% B (mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The mass spectrometer used a cone voltage of 37 V, a scan time of 0.5 s, and a mass/charge range of 50–1700 Tn.

To identify the peptide residues of hMUC16.nub with which H4sH8767P, H1H8794P2 and H1H8799P2 interact, LC-MSE data from the non-deuterated sample was processed and a database search was conducted that included sequences for hMUC16.nub, pepsin and randomized sequence using software Waters ProteinLynx Global Server (PLGS). The identified peptides were imported into DynamX software and filtered by two criteria: 1) minimum number of products per amino acid: 0.25 and 2) replication file threshold: 2. DynamX software then automatically determined the deuterium uptake of each peptide based on retention time and high mass accuracy (<10 ppm) at multiple time points with 3 replicates each time.

Using the online pepsin/protease XIII column in combination with MSE data acquisition, a total of 109 peptides from hMUC16.nub were identified in the absence or presence of H4sH8767P, representing 64% sequence coverage. Six peptides significantly reduced deuteration uptake (centroid delta values >0.5 Da from at least one time point with p -values <0.05) upon binding to H4sH8767P and are illustrated in Table 1 . 9A. The recorded peptide mass corresponds to the average of the MH ⁺ centroid mass from three replicates. These peptides, corresponding to amino acids 428-434, 453-467 and 474-481 of hMucl6.nub, had lower deuteration rates when bound to H4sH87 67P. These identified residues also correspond to residues 14237-14243, 14262-14276 and 14283-14290 of hMUC16, as defined in Uniprot entry Q8WXI7 (MUC16_HUMAN), SEQ ID NO: 1899.

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Table 9A

hMUC16.nub peptides with altered deuteration rates upon H4sH8767P binding

Leftovers SEQ ID NO: 1902 1 min. deuteration 20 minutes. deuteration Amino acid sequence hMUC 16.nub hMUC 16.nub +H4sH8767P Δ hMUC 16. nub hMUC 16.nub +H4sH8767P Δ 428-434 LYKGSQL 809.97 ± 0.03 809.07 ± 0.06 -0.26 811.05 ±0.16 810.17 ±0.01 -0.88 429-434 YKGSQL 697.10 ± 0.00 696.74 ± 0.00 -0.35 698.13 ±0.02 697.60 ± 0.03 -0.52 453-467 VTVKALFS SNLDPSL 1595.35 ± 0.08 1593.97 ±0.2 -1.38 1596.01 ±0.08 1595.33 ± 0.03 -0.68 459-467 FSSNLDPSL 983.19 ± 0.01 981.57 ±0.08 -1.62 983.51 ±0.03 982.76 ± 0.07 -0.75 460-467 SSNLDPSL 835.44 ± 0.01 834.01 ±0.00 -1.43 835.89 ±0.01 835.12 ±0.15 -0.74 474-481 DKTLNASF 899.76 ± 0.00 899.25 ± 0.06 -0.51 900.63 ±0.00 900.10 ±0.03 -0.54

Using the online pepsin/protease XIII column in combination with MS ^E data acquisition, a total of 109 peptides from hMUC16.nub were identified in the absence or presence of H1H8794P2, representing 64% sequence coverage. Three peptides significantly reduced deuteration uptake (centroid delta values >0.5 Da from at least one time point with p-values <0.05) when bound to H1H8794P2 and are illustrated in Table 1 . 9B. The recorded peptide mass corresponds to the average of the MH ⁺ centroid mass from three replicates. These peptides, corresponding to amino acids 126-131, 127-131 and 132-138 of hMucl6.nub, had lower deuteration rates when bound to H1H8794P2. These identified residues also correspond to residues 13935-13940, 13936-13940 and 13941-13947 of hMUC16, as defined in Uniprot entry Q8WXI7 (MUC16_HUMAN), SEQ ID NO: 1899.

Table 9B

hMUC16.nub peptides with altered deuteration rates upon binding of H1H8794P2

Leftovers SEQ ID NO: 1902 1 min. deuteration 20 minutes. deuteration Amino acid sequence hMUC 16.nub hMUC 16.nub ±H1H8794P 2 Δ hMUC 16. nub hMUC 16.nub +H1H8794P2 Δ 126-131 LRYMAD 771.41 ± 0.01 770.60 ± 0.04 -0.81 771.91 ±0.04 770.76 ± 0.02 -1.15 127-131 RYMAD 658.03 ± 0.02 657.32 ±0.01 -0.71 657.89 ±0.01 657.27 ±0.01 -0.6 132-138 MGQPGSL 692.55 ± 0.02 691.42 ±0.15 -1.13 692.61 ±0.01 691.58 ±0.02 -1.03

Using the online pepsin/protease XIII column in combination with MS ^E data acquisition, a total of 109 peptides from hMUC16.nub were identified in the absence or presence of H1H8799P2, representing 64% sequence coverage. Four peptides significantly reduced deuteration uptake (centroid delta values >0.5 Da from at least one time point with p-values <0.05) when bound to H1H8799P2 and are illustrated in Table 1 . 9C. The recorded peptide mass corresponds to the average of the MH ⁺ centroid mass from three replicates. These peptides, corresponding to amino acids 357-369, 358-366, 358-369 and 361-369 of hMucl6.nub, had lower rates of deuteration when bound to H1H8799P2. These identified residues also correspond to residues 14165-14178, 14166-14176, 14166-14178 and 14170-14178 of hMUC16, as op.

- 62 045730 allocated to Uniprot Q8WXI7 (MUC16_HUMAN), SEQ ID NO: 1899.

Table 9C

hMUC16.nub peptides with altered deuteration rates upon binding of H1H8799P2

Leftovers SEQ ID NO: 1902 1 min. deuteration 20 minutes. deuteration Amino acid sequence hMUC 16.nub hMUC 16.nub +Н1Н8799Р 2 Δ hMUC 16. nub hMUC 16.nub +H1H8799P2 Δ 357-369 LSQLTHGV TQLGF 1404.15 ± 0.03 1403.41 ± 0.09 -0.74 1406.14 ±0.15 1404.26 ± 0.02 -2,H 358-366 SQLTHGVT QL 972.37 ± 0.10 972.04 ±0.10 -0.33 973.94 ±0.03 972.56 ± 0.00 -1.38 358-369 SQLTHGVT QLGF 1291.23 ± 0.02 1290.20 ± 0.00 -1.03 1291.34 ±0.02 1291.05 ± 0.06 -2.27 361-369 THGVTQLG F 1404.15 ± 0.03 1403.42 ± 0.05 -0.73 1406.14 ±0.14 1404.03 ± 0.02 -2.11

Example 7. Pharmacokinetic evaluation of bispecific anti-MUC16χCD3 antibodies.

Evaluation of the pharmacokinetics of the anti-MUC16xCD3 bispecific antibodies BSMUC16/CD3-001 and BSMUC16/CD3-005 and isotype controls was performed in humanized MUC16xCD3 mice (mice homozygous for the expression of human MUC16 and CD3, MUC16 ^hu/hu x CD3 ^hu/hu ), humanized CD3 mice (mice homozygous to express human CD3, CD3 ^hu/hu ) and strain-matched wild-type (WT) mice (75% C57BL, 25% 129Sv). Cohorts contained 4-5 mice per antibody tested and per mouse strain. All mice received a single intraperitoneal (IP) dose of 0.4 mg/kg. Blood samples were collected at 3 and 6 hours, 1, 3, 7, 14 and 28 days after dosing. Blood was processed into serum and frozen at -80°C until analysis.

Circulating antibody concentrations were determined by a total human IgG antibody assay using GyroLab Xplore™ (Gyros, Uppsala, Sweden). Briefly, biotinylated goat polyclonal anti-human IgG antibody (Jackson ImmunoResearch, West Grove, PA) was captured onto streptavidin-coated beads on a Gyrolab Bioaffy 200 CD (Gyros) to capture human IgG present in the serum. After capture by the affinity column, bound human IgG antibody in the samples was detected using goat anti-human IgG labeled A1exa-647 (Jackson ImmunoResearch). The fluorescent signal on the column, detecting bound IgG, and response units (RU) were read by the instrument. Sample concentrations were determined by interpolation from a standard curve that was fitted using 5-parameter curve fitting using Gyrolab Evaluator software.

PK parameters were determined by noncompartmental analysis (NCA) using Phoenix® WinNonlin® software version 6.3 (Certara, LP, Princeton, NJ) and an extravascular dosing model. Using the corresponding average concentration values for each antibody, all PK parameters, including the observed maximum serum concentration ( _Cmax ), the estimated observed half-life (t1/ ₂ ) and the area under the concentration versus time curve to the last measured concentration (AUC _-nlast ) were determined using the linear trapezoidal rule with linear interpolation and uniform weighting.

Following intraperitoneal administration of antibodies to WT mice, total IgG concentration-time profiles of BSMUC16/CD3-001, BSMUC16/CD3-005, and isotype control were all similar, characterized by a first brief drug release phase followed by a single drug clearance phase for the remainder of the study. Maximum serum concentrations ( _Cmax ) and estimated drug exposure ( _AUC ) for the three _antibodies were comparable (1.3-fold of each other).

After intraperitoneal injection of CD3 ^hu/hu antibodies into mice, BSMUC16/CD3-001, BSMUC16/CD3005 and isotype control had comparable _Cmax concentrations (4.6, 3.6 and 4.1 μg/ml, respectively). BSMUC16/CD3-005 and the isotype control showed similar drug elimination curves, while BSMUC16/CD3-001 showed more dramatic drug elimination than both, suggesting that binding of the human CD3 target causes clearance. The final antibody concentration for BSMUC16/CD3-001 was 0.03 μg/ml, which is about 28 times less than the final antibody concentrations determined for the isotype control (0.85 μg/ml) and 22 times less than BSMUC16 /CD3-005 (0.66 μg/ml) serum concentrations.

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In MUC16 ^hu/hu x CD3 ^hu//hu doubly humanized mice, Muc16xCD3 bispecific and isotype control antibodies had comparable _Cmax concentrations ( _Cmax range: 4.5-6.9 μg/ml). Both bispecific antibodies showed higher drug elimination than the isotype control, suggesting a target-mediated effect. The antibody terminal concentrations for BSMUC16/CD3-001 and BSMUC16/CD3-005 were approximately 29-fold and 2.9-fold lower, respectively, than the antibody terminal concentrations determined for the isotype control (0.86 μg/ml).

A summary of data on total concentrations of anti-MUC16 x CD3 bispecific antibodies and isotype control antibodies is given in Table. 10. Average FC parameters are described in table. 11A and 11B. Mean total antibody concentrations over time are shown in FIG. 1, 2 and 3. In conclusion, bispecific MUC16xCD3 antibodies showed similar _Cmax and drug elimination curves in WT mice, but BSMUC16/CD3-001 showed higher elimination rates than BSMUC16/CD3-005 and isotype control in CD3 once humanized mice and MUC16/CD3 doubly humanized mice. Because the bispecific antibodies administered in this PK study consist of the same anti-MUC16 binding arm, the results suggest that the binding strength of the CD3-targeting arm may play a role in drug exposure levels (AUC) and drug clearance _rates . Neither BSMUC16/CD3-001 nor BSMUC16/CD3-005 binds mouse MUC16 or mouse CD3.

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Table 10

Mean serum total IgG concentrations following a single intraperitoneal injection of 0.4 mg/kg BSMUC16/CD3-001, BSMUC16/CD3-005, and isotype control antibodies in WT mice, humanized CD3 mice, and humanized MUC16 x CD3 mice

Antibody Time (D) Total mAb concentration in mouse serum DT CD3 ^hu/hu MUC16 ^hu/hu x CD3 ^hu/hu Average (µg/ml) +/-SD Average e (µg/ml) +/- SD Average (µg/ml) +/- SD BSMUC16/CD3- 001 0.13 5.39 0.34 4.30 0.29 6.77 1.52 0.25 5.80 0.36 4.26 1.07 6.63 1.06 1.00 4.13 0.43 2.87 0.71 4.89 0.53 3.00 3.19 0.53 1.44 0.27 2.50 0.22 7.00 2.61 0.73 0.72 0.13 1.20 0.22 14.00 1.44 0.69 0.18 0.05 0.28 0.08 21.00 0.93 Not detected 0.07 0.02 0.06 0.05 28.00 0.60 Not detected 0.04 0.01 0.03 0.02 BSMUC16/CD3- 005 0.13 4.23 0.62 3.35 1.15 4.35 0.24 0.25 4.53 0.55 3.40 0.96 4.45 0.49 1.00 3.47 0.32 2.72 0.42 3.00 0.61 3.00 2.51 0.13 1.95 0.37 1.98 0.41 7.00 2.02 0.24 2.31 0.67 1.58 0.36 14.00 1D9 0.17 1.01 0.23 0.78 0.26 21.00 1.19 0.29 1.19 0.11 0.66 0.29 28.00 0.71 0.20 0.66 0.28 0.30 0.22 Isotypic control 0.13 5.07 1.16 5.43 1.30 6.56 0.70 0.25 5.91 1.10 5.67 1.91 6.48 0.90 1.00 2.64 0.24 2.98 1.14 2.82 0.30 3.00 2.05 0.06 2.29 0.83 1.57 0.37 7.00 1.80 0.25 2.14 0.85 1.96 0.37 14.00 1.22 0.28 1.48 0.66 1.34 0.37 21.00 1.20 0.58 1.43 0.72 1.24 0.44 28.00 0.73 0.24 0.85 0.29 0.86 0.41

Time: (hours, if noted) = time in hours after a single dose; D=day of training; 30=standard deviation; NA=not detected due to exclusion of mice with drug-clearing anti-drug titers.

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Table 11A

Summary of pharmacokinetic parameters: CD3 ^hu/hu humanized mice

Parameter Units Mice DT CD3 ^hu/hu mice Isotypic control BSMUC16/CD3-001 BSMUC16/CD3-005 Isotypic control BSMUC16/CD3-001 BSMUC16/ CD3-005 With ^max µg/ml 5±3 6 ±0.4 5 ±0.5 4.1 ±3 4.6 ±0.8 3.5 ± 1 Τι/ ₂ d 11 ±4 7±3 12 ±2 14 ±0.5 3.9 ±0.6 11 ±5 A U Last d µg/ml 35 ±18 40 ± 11 45 ±5 49 ±20 16 ±3 36 ± 13

Cmax=peak concentration; AUC=area under the concentration-time curve; AUC-nlast=AUC calculated from the time of zero to the time of the last positive concentration; T] _/2 = expected half-life.

Table 11B Summary of pharmacokinetic parameters:

_____________MUC16 ^hu/hu x CD3 ^hu/hu doubly humanized mouse_____________

Parameter Units Mice DT MUC16 ^hu/hu x CD3 ^hu/hu mice Isotypic control BSMUC16/CD3-001 BSMUC16/CD3-005 Isotypic control BSMUC16/CD3-001 BSMUC16/ CD3-005 With ^max µg/ml 5±3 6 ±0.4 5 ±0.5 6.7 ±0.7 6.9 ±1 4.5 ±4 Τι/ ₂ d 11 ±4 7±3 12 ±2 12.9 ±4 3.3 ±0.8 8.2 ±4 A U Last D □ µg/ml 35 ±18 40 ± 11 45 ±5 46 ± 10 27 ±3 34 ± 11

Cmax=peak concentration; AUC=narea under the concentration-time curve; AUC-nlast=AUC calculated from the time of zero to the time of the last positive concentration; T] _/2 = expected half-life.

Example 8 Anti-MUC16/anti-CD3 bispecific antibodies exhibit potent antitumor efficacy in vivo.

To determine the in vivo efficacy of exemplary anti-MUC16/anti-CD3 bispecific antibodies identified as having little or no detectable binding to human and cynomolgus CD3, studies were conducted in immunocompromised mice bearing human prostate cancer xenografts. The effectiveness of the selected bispecific antibodies was tested in both acute and therapeutic dosage treatment models.

Efficacy of anti-MUC16/anti-CD3 bispecific antibodies in human tumor xenograft models.

To evaluate the in vivo efficacy of bispecific anti-MUC16/anti-CD3 in human tumor xenograft studies, NOD scid gamma (NSG) mice (Jackson Laboratories, Bar Harbor, Maine) were pre-implanted with human peripheral blood mononuclear cells (PBMCs; ReachBio LLC., Seattle). , Washington) and then given ascites cells from the human ovarian cancer cell line OVCAR-3 (American Type Tissue Culture, Manassas, VA) transduced with luciferase (OVCAR-3/Luc). OVCAR-3 cells endogenously express MUC-16.

Briefly, NSG mice were injected intraperitoneally (i.p.) with 5.0 x 10 ⁶ human PBMSA. 8 days later, 1.5 x 106 ascites cells from the OVCAR-3/Luc cell line, previously cultured in vivo, were injected intraperitoneally into NSG mice implanted with PBMCs. In the immediate treatment group, mice were treated intraperitoneally on the day of OVCAR-3/Luc cell implantation with the bispecific MUC16/CD3 antibodies BSMUC16/CD3-001 or BSMUC16/CD3-005 or isotype control at a dose of 10 μg/mouse (N=5 mice/treatment group). . In the therapeutic dose model, mice were administered intraperitoneally. 7 days after tumor implantation with bispecific MUC16/CD3 or control antibodies described above at a dose of 10 μg/mouse (N=5/treatment group).

In all studies, tumor growth was monitored using bioluminescence imaging (BLI). Mice were injected intraperitoneally with the luciferase substrate D-luciferin suspended in PBS (150 mg/kg) and imaged under isoflurane anesthesia after 10 min. BLI was performed using the Xenogen IVIS system (Perkin Elmer, Hopkinton, MA), and BLI signals were extracted using Living Image software (Xenogen/Perkin Elmer). Regions of interest were drawn around each cell mass and photon intensities were recorded as photons(ph)/sec(s)/ ^cm2 /steradian(sr). For the immediate treatment group, data are presented as BLI levels 26 days after tumor implantation (Table 12A). For the group receiving therapeutic

- 66 045730 treatment, data are presented as fold change in BLI between day 6 (1 day before treatment) and the study end point (day 26 after tumor implantation; Table 12B).

As the results show, both BSMUC16/CD3-001 and BSMUC16/CD3-005 showed equal efficacy in suppressing tumor growth compared to isotype control when BLI was measured at day 26 in the immediate dosing model. Both bispecific anti-MUC16/αanti-CD3 antibodies also suppressed the growth of established tumors when administered 7 days after tumor implantation compared to controls. Thus, the bispecific anti-MUC16/anti-CD3 antibodies of the present invention demonstrate strong antitumor efficacy in several models.

Table 12A

Efficacy of bispecific anti-MUC16/anti-CD3 antibodies in an immunocompromised xenograft model: immediate dosing

Tumor model/mouse line/Dose Bispecific Antibody ID N Wed. bioluminescent emission (photons/sec/ ^cm2 /steradian) day 26 (mean ± SD) OVCAR-3/Luc/ NSG/ 10 µg/mouse BSMUC16/CD3-001 5 1.4x10 ³ ± 3.5x10 ² [01] BSMUC16/CD3-005 5 1.5x10 ³ ± 9.7x10 ² [01] Isotypic control 5 2, OxiO ⁷ ± 1, OxO ⁶

Table 12B

Efficacy of bispecific anti-Muc16/anti-CD3 antibodies in an immunocompromised xenograft model: therapeutic treatment

Tumor Model/Mouse Line/Dose Bispecific Antibody ID N Multiplicity of change in Avg. bioluminescent radiation (f/sec/ ^cm2 /st) on day 26 relative to day 6 (mean ± SD) OVCAR-3/Luc/ NSG/ 10 µg/mouse BSMUC16/CD3-001 5 2.0 + 5.0 [01] BSMUC16/CD3-005 5 0.01 + 0.02 [01] Isotypic control 5 21.0 ± 8.0

In further experiments, the in vivo efficacy of the bispecific anti-MUC16/anti-CD3 antibody was assessed in xenogeneic and syngeneic tumor models. For the first xenogeneic model, NSG mice were intraperitoneally (ip) injected with OVCAR-3/Luc cells pre-passenged in vivo (day 0) eleven days after engraftment of human PBMCs. Mice were treated intraperitoneally with 0.01, 0.1, or 0.5 mg/kg BSMUC16/CD3-001 or treated with 0.5 mg/kg nonbinding control or CD3-binding control on days 6, 10, 13, 16, and 21. Tumor weight was assessed using BLI on days 6, 14, and 20 after tumor implantation. Treatment with 0.1 or 0.5 mg/kg BSMUC16/CD3-001 resulted in significant antitumor efficacy as determined by BLI measurements at day 20, as shown in Table. 13A-C and FIG. 4-6. For the second xenogeneic model, NSG mice were injected with OVCAR-3/Luc cells pre-passenged in vivo (day 0) thirteen days after engraftment of human PBMCs, and a second group of PBMCs were transferred on day 4. Mice were treated intravenously (IV) with 0.1, 0.5, 1, or 5 mg/kg BSMUC16/CD3-005 or administered 5 mg/kg nonbinding control or CD3 binding control on days 5, 8, 12, 15 , 19 and 22. Tumor burden was assessed using BLI on days 4, 11, 18 and 25. Treatment with 0.5, 1 or 5 mg/kg BSMUC16/CD3-005 given

- 67 045730 led to significant antitumor efficacy, as shown by measurements and fold change in BLI (Table 13D-F and Figs. 7-9). To investigate efficacy in an immunocompetent model, the mouse CD3 gene was replaced with human CD3, and part of the mouse MUC16 gene was replaced with human sequence. The replacement resulted in a mouse whose T cells express human CD3 and which expresses a chimeric MUC16 molecule containing the portion of human MUC16 where the bispecific BSMUC16/CD3-001 antibody binds. For the syngeneic tumor model, ID8-VEGF cell lines engineered to express a portion of human MUC16 were used. Mice were implanted with ID8-VEGF/huMUC16 cells subcutaneously and treated with 100 μg of BSMUC16/CD3-001 either on the day of implantation or ten days after implantation when tumors were detected. Treatment with 100 μg of BSMUC16/CD3-001 resulted in significant antitumor efficacy, as shown in Table. 13G and in FIG. 10.

Implantation and measurement of tumor xenografts: Ascites cells from the OVCAR-3/Luc cell line, previously passengered in vivo, were injected intraperitoneally into NSG mice previously implanted with human PBMCs. BLI was measured as a reading of tumor growth several days after OVCAR-3/Luc implantation and several times during the study. After the initial BLI measurement for cohorting, mice were divided into groups of 4-6 animals each and administered bispecific MUC16xCD3 or control antibodies twice weekly for the duration of the study.

Calculation of tumor growth and inhibition.

xenograft: Bioluminescence imaging was used to measure tumor burden. Mice were injected intraperitoneally with 150 mg/kg (as determined by body weight at the start of the experiment) of the luciferase substrate D-luciferin suspended in PBS. Ten minutes after dosing, BLI imaging was performed in mice under isoflurane anesthesia using the Xenogen IVIS system. Image acquisition was carried out with a field of view in D, an object height of 1.5 cm and an average binning level for 0.5 min of exposure time. BLI signals were acquired using Living Image software. Regions of interest were drawn around each tumor mass and photon intensities were recorded as f/s/cm ² /sr. Statistical analysis was performed using GraphPad Prism software (version 6). Statistical significance of BLI results was determined using an unpaired nonparametric Mann-Whitney t test. The fold change was calculated using the formula: (Day 20-Day 6)/Day 6 for Study 1 and (Day 25-Day 4)/Day 4 for Study 2.

Implantation and measurement of syngeneic tumors: Mice expressing human CD3 and human-mouse chimera MUC16 at the corresponding mouse loci were implanted with 10e6 ID8VEGF/huMUC16 cells subcutaneously (s.c.). Mice were administered BSMUC16/CD3-001 or CD3-binding control i.p. twice weekly for the duration of the study. Treatment began on day 0 or day 10 after implantation. Tumor growth was measured with a caliper twice a week. Mice were sacrificed 47 days after tumor implantation.

Calculation of syngeneic tumor growth and inhibition: To determine tumor volume, the largest longitudinal diameter (length) and largest transverse diameter (width) were determined using an external caliper. Tumor volume based on measurements with calipers was calculated using the formula: volume = (length x width ² )/2. Statistical significance was determined using an unpaired nonparametric Mann-Whitney t test.

The antitumor efficacy of the bispecific BSMUC16/CD3-001 antibody in xenogeneic and syngeneic in vivo tumor models is shown in Table. 13A-D, below.

Table 13A

Model Study 1 OVCAR-3. Bioluminescence level on day 6 after tumor implantation

Antibody (mg/kg) Wed. radiation [f/sec/ ^cm2 /st] 6 days after implantation (median ± SEM) non-binding control (0.5) 8.15e+05 ± 7.88 e + 04 CD3 binding control (0.5) 6.3 9e + 05 ± 8.67e+04 BSMUC16/CD3-001 (0.5) 7.64e+05 ± 1.19 e + 05 BSMUC16/CD3-001 (0.1) 6.31e+05 ± 1.10 e+05 BSMUC16/CD3-001 (0.01) 8.77e+05 ± 7.91 e+04

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Table 13B

Model Study 1 OVCAR-3. Bioluminescence level on day 20 after tumor implantation

Antibody (mg/kg) Wed. radiation [f/sec/ ^cm2 /st] 20 days after implantation (median ± SEM) non-binding control (0.5) 8.63е+0 6 + 1.45 e + 06 CD3 binding control (0.5) 9.94e + 0 6 ± 1.08e+06 BSMUC16/CD3-001 (0.5) 9.37e+02 ± 9.62 e+02 BSMUC16/CD3-001 (0.1) 2.36e+04 ± 1.28 e+06 BSMUC16/CD3-001 (0.01) 6.51e+06 ± 1.60 e + 06

Table 13C

Model Study 1 OVCAR-3. Fold change in BLI between 6 and 20 days after tumor implantation

Antibody (mg/kg) Multiplicity of change in avg. radiation [f/sec/cm ² /st] from 6 days to 20 days after implantation (mean ± SD) non-binding control (0.5) 9.5 ± 19 CD3 binding control (0.5) 15.6 ± 6.7 BSMUC16/CD3-001 (0.5) -1.00 ± 0.00 BSMUC16/CD3-001 (0.1) 1.2 ± 4.7 BSMUC16/CD3-001 (0.01) 5, 6 ± 4.2

Table 13D

Model Study 2 OVCAR-3. Bioluminescence level on day 4 after tumor implantation

Antibody (mg/kg) Wed. radiation [f/sec/ ^cm2 /st] 4 days after implantation (median + SEM) non-binding control (5) 1.54e+05 ± 9.93e+03 CD3 binding control (5) 1.34e+05 ± 1.55e+04 BSMUC16/CD3-005 (5) 1.54e+05 ± 1.03e+04 BSMUC16/CD3-005 (1) 1.38e+05 ± 4.65e+03 BSMUC16/CD3-005 (0.5) 1.31e+05 ± 4.03e+03 BSMUC16/CD3-005 (0.1) 1.53e+05 ± 1.93e+04

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Table 13E

Model Study 2 OVCAR-3. Bioluminescence level on day 25 after tumor implantation

Antibody (mg/kg) Wed. radiation [f/sec/ ^cm2 /st] 25 days after implantation (median ± SEM) non-binding control (5) 7.20e+06 ± 8.91e+05 CD3 binding control (5) 6.15e+06 ± 7.26e+05 BSMUC16/CD3-005 (5) 1.52e+03 ± 4.86e+05 BSMUC16/CD3-005 (1) 6.99е+03 ± 6.23е+03 BSMUC16/CD3-005 (0.5) 2.23e+03 ± 2.35e+05 BSMUC16/CD3-005 (0.1) 7,bЗе+06 ± 1.83е+06

Table 13F

Model Study 2 OVCAR-3. Fold change in BLI between 4 and 25 days after tumor implantation

Antibody (mg/kg) Multiplicity of change in avg. radiation [f/sec/ ^cm2 /st] from 4 days to 25 days after implantation (mean ± SD) non-binding control (5) 46.8 ± 20.6 CD3 binding control (5) 55.0 ± 14.7 BSMUC16/CD3-005 (5) 2.5 ± 8.5 BSMUC16/CD3-005 (1) -0.9 ± 0.1 BSMUC16/CD3-005 (0.5) 0.7 + 3.6 BSMUC16/CD3-005 (0.1) 45.4 ± 35.7

Table 13G

Model ID8-VEGF/huMUC16. Tumor size (mm ³ ) on day 47

Start of treatment Antibody (mcg) Tumor size ( ^mm3 ) at day 47 (mean ± SEM) Day 0 CD3 binding control (100) 827.5 ± 223.5 Day 0 BSMUC16/CD3-001 (100) 51.2 ± 51.2 Day 10 BSMUC16/CD3-001 (100) 273.8 ± 92.36

Example 9. Preparation and characterization of the conjugate.

All monoclonal antibodies were expressed in CHO cells and purified with protein A. An isotype control was also prepared in a similar manner. The non-binding isotype control antibody was derived from an immunological antigen not related to oncology.

Antibody (10 mg/ml) in 50 mM HEPES, 150 mM NaCl, pH 7.5 was treated with 1 mM dithiothreitol at 37°C for 30 min. After gel filtration (G-25, pH 4.5 sodium acetate), one of the maleimido linker payload derivatives of Compound 7 or Compound 10 (see Table 14) was added to the DMSO derivative (10 mg/ml) (1.2 equivalents /SH group) in DMSO (10 mg/ml) to reduce antibody content, and the mixture was adjusted to pH 7.0 with 1 M HEPES (pH 7.4). Compound 7 and Compound 10, as well as methods for preparing the compounds, are described in PCT Publication No. WO2014/145090, published September 18, 2014, and PCT Publication No. WO2016/160615, published October 6, 2016, respectively, each of which is included in its entirety in This description is for reference only. After 1 h, the reaction was quenched with an excess of N-ethylmaleimide. Conjugates were purified by size exclusion chromatography

- 70 045730 sizes and sterile filtered. Protein and linker concentrations were determined using UV spectral analysis. Size exclusion HPLC determined that all conjugates used were >95% monomeric. The outputs are given in table. 14 based on protein determination. All conjugated antibodies were analyzed by UV light for linker payload loading values according to Hamblett et al., Cancer Res 2004 10 7063. The results are summarized in table. 14.

A conjugate containing compound 60 can be prepared using a similar method. Compound 60 and methods for preparing the compound are described in PCT Publication No. WO2016/160615 (Example 20), published October 6, 2016, which is incorporated herein by reference in its entirety. Compound 60 is maytansine-N-methyl-L-alanine-(3-methoxy-4amino)benzamido-Cit-Val-Cap-Mal.

Table 14

Summary of payload (chemotoxic drug) and antibody-drug-conjugate parameters

Compound ε252 nm (cm ^-1 M ¹ ) ε280 nm (cm ^-1 M ¹ ) 7 [Maytansin-3-N-methyl-L-(S) -alanine-propanamidyl-3-N- methyl-N-[4- (amino-citrulline-valine- hexanamide-6- maleimidyl)benzyl]carbamate] 50600 8100 10[Maytansine-N-methyl-Lalanine-4-aminobenzamidecitrulline-valine-caprolyl-6maleimidyl] 45990 20600 Antibody ε252 nm (cm ^-1 M ¹ ) ε280 nm (cm ^-1 M ¹ ) H1H9519N 83995 235280 H1H9521N 85564 232050 Isotype control 75113 218360 Antibody conjugate Useful load: antibody (UV) Productivity % H1H9519N-7 3.5 40 H1H9521N-7 3.6 40 H1H9521N-1O 3.0 40 Isotype control-7 3.0 60 Isotype control-10 3.4 60

Example 10 Anti-MUC16 antibody drug conjugates are potent tumor growth inhibitors in in vivo MUC16-positive prostate cancer xenograft models.

To determine the in vivo efficacy of anti-MUC16 antibodies conjugated to Compound 7 and Compound 10, studies were conducted in immunocompromised mice bearing MUC16 positive ovarian cancer xenografts.

For these studies, female SCID mice (Taconic, Hudson, NY) were implanted with OVCAR3 [NIH:OVCAR3 (OVCAR3, ATCC NTB-161)] cells transfected with luciferase (OVCAR3/luc), which endogenously express MUC16. For intraperitoneal (IP) tumors, mice were randomized to treatment groups and treated with either drug-conjugated anti-MUC16 antibody (see Example 9), non-binding conjugated antibody, or vehicle upon detection of tumor luminescent signal. For subcutaneous (SC) xenografts, after tumors reached a mean volume of 200 mm ³ (~day 16), mice were randomized into treatment groups and received either drug-conjugated anti-MUC16 antibody, non-binding drug-conjugated antibody, or carrier. In these in vivo studies, antibodies

- 71 045730 were treated and tumors were then monitored until the development of ascites or an average tumor size of approximately 1200 mm ³ in the vehicle-only group. At this point, tumor growth inhibition was calculated.

In an initial intraperitoneal (IP) study, as an example, anti-MUC16 antibodies conjugated to compound 7 were tested for effectiveness in reducing OVCAR3/luc luminescence signal. Mice received four doses of anti-MUC16 and control ADCs once weekly at 85 μg/kg drug equivalent based on the ADC drug:antibody ratio. As shown in table. 15A, H1H9519N-compound 7 and H1H9521N-compound-7 strongly inhibit ascites tumor growth. These anti-MUC16 ADCs were effective in reducing tumor size with a 100% reduction in tumor fluorescence compared to vehicle control. The control ADC did not mediate any inhibition by OVCAR/luc of ascitic tumor cell growth.

Further study evaluating the efficacy of anti-MUC16 ADC against subcutaneous (SC) OVCAR3/luc tumor is summarized in Table. 15V. Mice received four doses of anti-MUC16 and control ADCs once weekly at 85 μg/kg drug equivalent based on the ADC drug:antibody ratio. When conjugated to compound 7, MUC16 ADC H1H9519N Compound 7 and H1H9521N-Compound 7 again exerted significant antitumor effects; this time against subcutaneous OVCAR3/luc tumors. Accordingly, these ADCs mediated 100% and 109% tumor growth inhibition, respectively. The control ADC did not mediate any inhibition of subcutaneous OVCAR/luc tumor growth.

In the third study, the efficacy of the H1H9521N anti-MiC16 antibody conjugated to the drug linker compound 10 was assessed in the IP OVCAR3/luc tumor model. Mice received single doses of anti-MiC16 and control ADCs at 85 μg/kg, 170 μg/kg, and 340 μg/kg drug equivalents based on the ADC drug:antibody ratio. As shown in table. 15C, H1H9519N-10 strongly inhibits ascites tumor growth. Doses of H1H9519N-compound 10 resulted in 99-100% inhibition of tumor fluorescence relative to vehicle control. Some inhibition was observed for the control ADC using compound 10, although it was more modest than that observed after anti-MiC16 H1H9519N-compound 10.

Table 15A

IP inhibition of OVCAR3/luc tumor growth on day 49 in SCID mice treated with anti-MUC16 antibodies conjugated to compound 7

Treatment group Average final tumor radiation Average tumor growth inhibition (%) (mean ± SEM) Carrier 16469750 ± 10679335 0 Control compound 7 85 µg/kg 16813750 ± 4026065 -2 H1H9519N- Compound 7 85 µg/kg 111254 ± 187288 100 H1H9521N- Compound 7 85 µg/kg 110413 ± 161353 100

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Table 15B

SC inhibition of OVCAR3/luc tumor growth on day 37 in SCID mice treated with anti-MUC16 antibodies conjugated to compound 7

Treatment group Final tumor volume (mean ± SEM) Average tumor growth inhibition (%) Carrier 1210 ± 426 0 Control-Compound 7 85 µg/kg 1737 ± 391 -51 H1H9519N- Connection 7 85 µg/kg 187 ± 269 100 H1H9521N- Connection 7 85 µg/kg 89 ± 97 109

Table 15C

IP inhibition of OVCAR3/luc tumor growth on day 49 in SCID mice treated with anti-MUC16 antibodies conjugated to compound 10

Treatment group Final tumor radiation (mean ± SEM) Average tumor growth inhibition (%) Carrier 29211000 ± 23504780 0 Control-Connection 10 85 mcg/kg 17332625 ± 14346694 41 Control-Connection 10 170 mcg/kg 32075000 ± 15623403 -10 Control-Connection 10 340 mcg/kg 22882350 ± 18771913 22 H1H9521N- Connection 10 85 mcg/kg 574285 ± 306844 99 H1H9521N- Connection 10 170 mcg/kg 236037 ± 226948 100 H1H9521N- Connection 10 340 mcg/kg 26472 ± 25079 101

Example 11. Preparation of anti-CD3 antibodies.

Ahtu-CD3 antibodies are produced by immunizing an engineered mouse containing DNA encoding human immunoglobulin heavy and kappa light chain variable regions with cells expressing CD3 or with DNA encoding CD3. The antibody response was monitored using a CD3-specific immunoassay. Once the desired immune response was achieved, splenocytes were collected and fused with mouse myeloma cells to maintain their viability and generate hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines that produce CD3-specific antibodies. Using this technique, several chimeric anti-CD3 antibodies (ie, antibodies having human variable domains and mouse constant domains) have been produced. In addition, several fully human anti-CD3 antibodies have been isolated directly from antigen-positive B cells without fusion with myeloma cells, as described in US 2007/0280945A1.

Certain biological properties of exemplary anti-MUC16 antibodies produced in accordance with the methods of this example are described in detail in the examples provided herein.

Example 12. Sequences of amino acids and nucleic acids of the variable region of the heavy and light chains.

In table 16 shows the amino acid sequence identifiers of the variable regions and heavy and light chain CDRs of selected anti-MUC16 antibodies according to this invention. Correspondence

- 73 045730 The current nucleic acid sequence identifiers are given in table. 17. Methods for producing the anti-CD3 antibodies described herein can also be found in US Publication No. 2014/0088295.

Table 16 Amino acid sequence identifiers

SEQ ID NO: Antibody designation HCVR HCDR1 HCDR3 LCVR LCDR11 LCDR2 LCDR3 H1H2712N 402 404 406 408 410 412 414 416 H1M2692N 418 420 422 424 426 428 430 432 H1M3542N 434 436 438 440 442 444 446 448 H1M3544N 450 452 454 456 458 4 60 4 62 4 64 H1M3549N 466 4 68 470 472 474 476 478 480 H1M3613N 482 484 486 488 490 492 494 496 H2M2689N 498 500 502 504 506 508 510 512 H2M2690N 514 516 518 520 522 524 52 6 528 H2M2691N 530 532 534 53 6 538 540 542 544 H2M2704N 546 548 550 552 554 556 558 560 H2M2705N 562 564 566 568 570 572 574 576 H2M2706N 578 580 582 584 586 588 590 592 H2M2707N 594 596 598 600 602 604 60 6 608 H2M2708N 610 612 614 616 618 620 622 624 H2M2709N 62 6 628 630 632 634 636 638 64 0 H2M2710N 642 64 4 64 6 648 650 652 654 65 6 H2M2711N 658 660 662 664 666 668 670 672 H2M2774N 67 4 676 678 680 682 684 68 6 688 H2M2775N 690 692 694 69 6 698 700 702 704 H2M2776N 706 708 710 712 714 716 718 720 H2M2777N 722 724 726 728 730 732 734 73 6 H2M2778N 738 740 742 744 746 748 750 752 H2M2779N 754 75 6 758 760 762 764 766 768 H2M2789N 770 772 774 776 7 78 780 782 784 H2M2862N 78 6 788 790 792 794 796 798 800 H2M2885N 802 804 806 808 810 812 814 816 H2M2886N 818 820 822 824 826 828 830 832 H2M3540N 834 836 838 840 842 844 846 848 H2M3541N 850 852 854 856 858 8 60 8 62 8 64 H2M3543N 866 8 68 870 872 874 876 878 880 H2M3547N 882 884 886 888 890 892 894 896 H2M3548N 898 900 902 904 906 908 910 912 H2M3563N 914 916 918 920 922 924 92 6 928 Н1Н5751Р 930 932 934 93 6 938 940 942 944

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H1H5752P 946 948 950 952 954 956 958 960 H1H5753B 962 964 966 968 970 972 974 976 H1H5754B 978 980 982 984 986 988 990 992 H1H5755B 994 996 998 1000 1002 1004 1006 1008 H1H5756B 1010 1012 1014 1016 1018 1020 1022 1024 H1H5757B 102 6 1028 1030 1032 1034 103 6 1038 1040 H1H5758B 1042 1044 1046 1048 1050 1052 1054 1056 H1H5761P 1058 10 60 1062 1064 1066 1068 1070 10 72 H1H5763P 1074 1076 1078 1080 1082 1084 1086 1088 H1H5764P 1090 1092 1094 1096 1098 1100 1102 1104 H1H5769P 1106 1108 1110 1112 1114 1116 1118 1120 H1H5771P 1122 1124 1126 1128 ISO 1132 1134 1136 H1H5772P 1138 1140 1142 1144 1146 1148 1150 1152 H1H5777P 1154 1156 1158 1160 1162 1164 1166 1168 H1H5778P 1170 1172 1174 1176 1178 1180 1182 1184 H1H5780P 1186 1188 1190 1192 1194 1196 1198 1200 H1H5781P 1202 1204 1206 1208 1210 1212 1214 1216 H1H5782P 1218 1220 1222 1224 1226 1228 1230 1232 H1H5785B 1234 1236 1238 1240 1242 1244 1246 1248 H1H5786B 1250 1252 1254 1256 1258 1260 1262 1264 H1H5788P 1266 12 68 1270 1272 1274 1276 1278 1280 H1H5790B 1282 1284 1286 1288 1290 1292 1294 1296 H1H5791B 1298 1300 1302 1304 1306 1308 1310 1312 H1H5792B 1314 1316 1318 1320 1322 1324 1326 1328 H1H5793B 1330 1332 1334 1336 1338 1340 1342 1344 H1H5795B 1346 1348 1350 1352 1354 1356 1358 1360 H1H5796B 1362 1364 1366 1368 1370 1372 1374 1376 H1H5797B 1378 1380 1382 1384 1386 1388 1390 1392 H1H5798B 1394 1396 1398 1400 1402 1404 1406 1408 H1H5799P 1410 1412 1414 1416 1418 1420 1422 1424 H1H5801B 1426 1428 1430 1432 1434 1436 1438 1440 H1H7194B 1442 1444 1446 1448 1634 163 6 1638 1640 H1H7195B 1450 1452 1454 1456 1634 163 6 1638 1640 H1H7196B 1458 14 60 14 62 14 64 1634 163 6 1638 1640 H1H7198B 1466 14 68 1470 1472 1634 163 6 1638 1640 H1H7203B 1474 1476 1478 1480 1634 163 6 1638 1640 H1H7204B 1482 1484 1486 1488 1634 163 6 1638 1640

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Н1Н7208В 1490 1492 1494 1496 1634 163 6 1638 1640 Н1Н7211В 1498 1500 1502 1504 1634 163 6 1638 1640 Н1Н7221В 1506 1508 1510 1512 1634 163 6 1638 1640 Н1Н7223В 1514 1516 1518 1520 1634 163 6 1638 1640 Н1Н7226В 1522 1524 1526 1528 1634 163 6 1638 1640 Н1Н7232В 1530 1532 1534 1536 1634 163 6 1638 1640 Н1Н7233В 1538 1540 1542 1544 1634 163 6 1638 1640 Н1Н7241В 1546 1548 1550 1552 1634 163 6 1638 1640 Н1Н7242В 1554 1556 1558 1560 1634 163 6 1638 1640 Н1Н7250В 1562 1564 1566 1568 1634 163 6 1638 1640 Н1Н7251В 1570 1572 1574 1576 1634 163 6 1638 1640 Н1Н7254В 1578 1580 1582 1584 1634 163 6 1638 1640 Н1Н7258В 1586 1588 1590 1592 1634 163 6 1638 1640 Н1Н7269В 1594 1596 1598 1600 1634 163 6 1638 1640 Н1Н7279В 1602 1604 160 6 1608 1634 163 6 1638 1640 H1XH7221G 1610 1612 1614 1616 1634 163 6 1638 1640 H1XH7221G3 1618 1620 1622 1624 1634 163 6 1638 1640 H1XH7221G5 162 6 1628 1630 1632 1634 163 6 1638 1640

Table 17

Nucleic acid sequence identifiers

SEQ ID NO: Antibody designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H2712N 401 403 405 407 409 411 413 415 H1M2692N 41 7 419 421 423 425 427 429 431 H1M3542N 433 435 437 439 441 443 445 447 H1M3544N 449 451 453 455 457 459 4 61 4 63 H1M3549N 4 65 467 4 69 471 473 475 477 479 H1M3613N 481 483 485 487 489 491 493 495 H2M2689N 497 499 501 503 505 507 509 511 H2M2690N 513 515 517 519 521 523 525 527 H2M2691N 529 531 533 535 537 539 541 543 H2M2704N 545 547 549 551 553 555 557 559 H2M2705N 561 563 5 65 567 5 69 571 573 575 H2M2706N 577 579 581 583 585 587 589 591 H2M2707N 593 595 597 599 601 603 605 60 7

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H2M2708N 609 611 613 615 617 619 621 623 H2M2709N 625 627 629 631 633 635 63 7 639 H2M2710N 641 643 645 647 649 651 653 655 H2M2711N 65 7 659 661 663 665 667 669 671 H2M2774N 673 675 677 679 681 683 685 68 7 H2M2775N 689 691 693 695 697 699 701 703 H2M2776N 705 707 709 711 713 715 717 719 H2M2777N 721 723 725 727 729 731 733 735 H2M2778N 737 739 741 743 745 747 749 751 H2M2779N 753 755 757 759 761 763 765 767 H2M2789N 769 771 773 7 75 777 779 781 783 H2M2862N 785 787 789 791 793 795 797 799 H2M2885N 801 803 805 807 809 811 813 815 H2M2886N 81 7 819 821 823 825 827 829 831 H2M3540N 833 835 837 839 841 843 845 847 H2M3541N 849 851 853 855 857 859 8 61 8 63 H2M3543N 865 867 8 69 871 873 875 877 879 H2M3547N 881 883 885 887 889 891 893 895 H2M3548N 897 899 901 903 905 907 909 911 H2M3563N 913 915 917 919 921 923 925 927 H1H5751P 929 931 933 935 937 939 941 943 H1H5752P 945 947 949 951 953 955 957 959 H1H5753B 961 963 9 65 967 9 69 971 973 975 H1H5754B 977 979 981 983 985 987 989 991 H1H5755B 993 995 997 999 1001 1003 1005 1007 H1H5756B 1009 1011 1013 1015 1017 1019 1021 1023 H1H5757B 1025 1027 1029 1031 1033 1035 1037 1039 H1H5758B 1041 1043 1045 1047 1049 1051 1053 1055 H1H5761P 1057 1059 10 61 10 63 1065 1067 10 69 1071 H1H5763P 1073 1075 1077 10 79 1081 1083 1085 1087 H1H5764P 1089 1091 1093 1095 1097 1099 1101 1103 H1H5769P 1105 1107 1109 1111 1113 1115 1117 1119 H1H5771P 1121 1123 1125 1127 1129 1131 1133 1135 H1H5772P 1137 1139 1141 1143 1145 1147 1149 1151

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H1H5777P 1153 1155 1157 1159 1161 1163 1165 1167 H1H5778P 1169 11 71 1173 1175 1177 1179 1181 1183 H1H5780P 1185 1187 1189 1191 1193 1195 1197 1199 H1H5781P 1201 1203 1205 1207 1209 1211 1213 1215 H1H5782P 121 7 1219 1221 1223 1225 1227 1229 1231 H1H5785B 1233 1235 1237 1239 1241 1243 1245 1247 H1H5786B 1249 1251 1253 1255 1257 1259 1261 1263 H1H5788P 1265 1267 12 69 1271 1273 1275 1277 1279 H1H5790B 1281 1283 1285 1287 1289 1291 1293 1295 H1H5791B 1297 1299 1301 1303 1305 1307 1309 1311 H1H5792B 1313 1315 1317 1319 1321 1323 1325 1327 H1H5793B 1329 1331 1333 1335 1337 1339 1341 1343 H1H5795B 1345 1347 1349 1351 1353 1355 1357 1359 H1H5796B 1361 1363 13 65 1367 1369 1371 1373 1375 H1H5797B 1377 1379 1381 1383 1385 1387 1389 1391 H1H5798B 1393 1395 1397 1399 1401 14 03 14 05 1407 H1H5799P 1409 1411 1413 1415 1417 1419 1421 1423 H1H5801B 1425 1427 1429 1431 1433 1435 1437 1439 H1H7194B 1441 1443 1445 1447 1633 1635 163 7 1639 H1H7195B 1449 1451 1453 1455 1633 1635 163 7 1639 H1H7196B 1457 1459 14 61 14 63 1633 1635 163 7 1639 H1H7198B 14 65 1467 14 69 1471 1633 1635 163 7 1639 H1H7203B 1473 1475 1477 1479 1633 1635 163 7 1639 H1H7204B 1481 1483 1485 1487 1633 1635 163 7 1639 H1H7208B 1489 1491 1493 1495 1633 1635 163 7 1639 H1H7211B 1497 1499 1501 1503 1633 1635 163 7 1639 H1H7221B 1505 1507 1509 1511 1633 1635 163 7 1639 H1H7223B 1513 1515 1517 1519 1633 1635 163 7 1639 H1H7226B 1521 1523 1525 1527 1633 1635 163 7 1639 H1H7232B 1529 1531 1533 1535 1633 1635 163 7 1639 H1H7233B 1537 1539 1541 1543 1633 1635 163 7 1639 H1H7241B 1545 1547 1549 1551 1633 1635 1637 1639 H1H7242B 1553 1555 1557 1559 1633 1635 163 7 1639 H1H7250B 1561 1563 15 65 1567 1633 1635 163 7 1639

H1H7251B 1569 1571 1573 1575 1633 1635 163 7 1639 H1H7254B 1577 1579 1581 1583 1633 1635 163 7 1639 H1H7258B 1585 1587 1589 1591 1633 1635 163 7 1639 H1H7269B 1593 1595 1597 1599 1633 1635 163 7 1639 H1H7279B 1601 1603 1605 160 7 1633 1635 163 7 1639 H1XH7221G 1609 1611 1613 1615 1633 1635 163 7 1639 H1XH7221G3 1617 1619 1621 1623 1633 1635 163 7 1639 H1XH7221G5 1625 162 7 1629 1631 1633 1635 163 7 1639

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Antibodies are generally referred to herein according to the following nomenclature: an Fc prefix (eg, H1H, H1M, H2M, etc.) followed by a numerical identifier (eg, 2712, 2692, etc., as shown in Table 1), followed by the suffix P, N, or B. Thus, according to this nomenclature, the antibody may be referred to herein as, for example, H1H2712N, H1M2692N, H2M2689N, etc. The prefixes H1H, H1M and H2M in the antibody designations used herein indicate the specific Fc region of the antibody isotype. For example, antibody H1H has a human IgG1 Fc, antibody H1M has a mouse IgG1 Fc, and antibody H2M has a mouse IgG2 Fc (all variable regions are fully human, as indicated by the first 'H' in the antibody designation). As one skilled in the art will appreciate, an antibody having a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., a mouse IgG1 Fc antibody can be converted to a human IgG4 antibody, and the like), but in either case , variable domains (including CDRs) - which are designated by numerical identifiers given in table. 1 will remain unchanged and the binding properties are expected to be identical or substantially the same regardless of the nature of the Fc domain.

Table 18 and 19 set out the amino acid sequence identifiers of the heavy chain variable regions (Table 18) and light chain variable regions (Table 19), and their corresponding CDRs, additional HCVR and LCVR anti-CD3, useful in anti-MUC16x anti-CD3 bispecific antibodies according to this invention.

Table 18

Amino acid sequences of the heavy chain variable region

SEQ ID NO: Heavy chain ID HCVR HCDR1 HCDR2 HCDR3 CD3-VH-AA 1642 1644 164 6 1648 CD3-VH-B 1658 1660 1662 1664 CD3-VH-C 1674 1676 1678 1680 CD3-VH-D 1690 1692 1694 1696 CD3-VH-E 1,706 1,708 1710 1,712 CD3-VH-F* 1721 1722 1723 1724

Table 19

Amino acid sequences of the light chain variable region

SEQ ID NO: Light chain ID LCVR LCDR1 LCDR2 LCDR3 CD3-VL-AA 1650 1652 1654 1656 CD3-VL-B 1666 1668 1670 1672 CD3-VL-C 1682 1684 1686 1688 CD3-VL-D 1698 1 700 1 702 1,704 CD3-VL-E 1714 1716 1718 1,720 CD3-VL-F* 1,725 1,726 1727 1,728

The heavy and light chain variable regions CD3-VH-F and CD3-VL-F were derived from the bhtu-CD3 antibody designated L2K as specified in WO 2004/106380.

In addition, in table. 20 and 21 set forth sequence identifiers for nucleotide sequences encoding heavy chain variable regions (Table 20) and light chain variable regions (Table 21), and their corresponding CDRs, additional anti-CD3 HCVR and LCVR useful in anti-MUC16 x anti-CD3 bispecific antibodies according to this invention.

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Table 20

Nucleotide sequences encoding heavy chain variable region sequences

SEQ ID NO: Heavy chain ID HCVR HCDR1 HCDR2 HCDR3 CD3-VH-AA 1641 1643 1645 1647 CD3-VH-B 1657 1659 1661 1663 CD3-VH-C 1673 1675 1677 1679 CD3-VH-D 1689 1691 1693 1695 CD3-VH-E 1705 1707 1709 1711

Table 21

Nucleotide sequences encoding light chain variable region sequences

SEQ ID NO: Light chain ID LCVR LCDR1 LCDR2 LCDR3 CD3-VL-AA 1649 1651 1653 1655 CD3-VL-B 1665 1667 1669 1671 CD3-VL-C 1681 1683 1685 1687 CD3-VL-D 1697 1699 1 701 1,703 CD3-VL-E 1,713 1,715 1717 1719

Control constructs used in the following examples.

Various control constructs (anti-CD3 antibodies) were included in the following experiments for comparative purposes: OKT-3, a mouse monoclonal antibody against human T cell surface antigens, is available from the American Type Culture Collection (ATCC) catalog no. CRL-8001; and SP34, a commercially available murine monoclonal antibody, obtained from, for example, Biolegend, San Diego, Calif. (Cat. No. 302914) or BD Pharmagen, Cat. 55052, reacts against the epsilon chain of the T3 complex on human T-lymphocyte cells.

Example 13. Preparation of additional anti-CD3 antibodies.

The following procedures were designed to identify antibodies that specifically recognize CD3 (a T cell co-receptor) as an antigen.

A pool of anti-CD3 antibodies was obtained from a genetically modified mouse. Briefly, mice were immunized with CD3 antigen and B cells were generated that contained multiple human VH rearrangements to express a diverse repertoire of high-affinity antigen-specific antibodies.

Antibodies described in table. 22-25, have the same light chain sequence VK1-39JK5 (LCVR presented in SEQ ID NO: 1890).

The generated antibodies were tested for binding to human and cynomolgus CD3 antigen in an in vitro binding assay, and, for example, one CD3 antibody was identified: designated CD3-VH-P (HCVR, represented in SEQ ID NO: 1882), among several others that have been found to bind to both human and cynomolgus CD3 having EC ₅₀ binding between 1 and 40 nM (or cell binding titration) as determined by FACS titrations of Jurkat cells and cynomolgus T cells, respectively; see also, for example, the FACS binding experiments described in Example 15 and PCT/US2016/044732, filed July 29, 2016.

The germline amino acid residues of CD3-VH-P were then identified (the V-D-J rearrangement for CD3-VH-P is IGHV3-9*01, IGHJ6*02, IGHD5-12*01) and the antibody designated CD3-VH-G was designed to contain germline scaffolds only. Other antibody derivatives have been developed using well-known molecular cloning techniques to replace amino acid residues in a stepwise manner based on differences between the germline sequence and the CD3-VH-P sequence. Each antibody derivative is given a CD3-VH-G designation number; see table 18.

While CD3-VH-G and some other engineered antibodies retain their binding as shown in FACS assays, several anti-CD3 antibodies are not related to human or Java CD3.

- 80 045730 macaque in vitro with weak or no measurable binding, e.g. 40 nM EC50. Binding affinity, binding kinetics and other biological properties for toxicity and pharmacokinetic (pK) profiles were subsequently examined for bispecific antibodies containing exemplary anti-CD3 antibodies prepared in accordance with the methods of this example, described in detail in the examples provided herein.

Example 14 Heavy and light chain variable regions (amino acid sequences and CDR nucleic acid sequences).

In table 22 shows the amino acid sequence identifiers of the variable regions and heavy chain CDRs of selected anti-MUC16 antibodies of this invention. The corresponding nucleic acid sequence identifiers are given in Table. 23.

Amino acid and nucleic acid sequences were determined for each antibody heavy chain sequence.

Each antibody heavy chain derived from the germline sequence (SEQ ID NO: 1910) was assigned a G designation for consistent nomenclature. In table 22 shows the amino acid sequence identifiers of the heavy chain variable regions and CDRs of the constructed anti-CD3 antibodies of the present invention. The corresponding nucleic acid sequence identifiers are given in Table. 23. The light chain variable region amino acid and nucleic acid sequence identifiers and CDRs are also listed below in Table. 24 and 25 respectively.

Table 22 Heavy chain amino acid sequence identifiers

Designation CD3-VH Antibodies SEQ ID NO: HCVR CDR1 CDR2 CDR3 CD3-VH-G 1,730 1,732 1,734 1,736 CD3-VH-G2 1,738 1,740 1,742 1,744 CD3-VH-G3 1,746 1,748 1,750 1,752 CD3-VH-G4 1,754 1,756 1,758 1,760 CD3-VH-G5 1,762 1,764 1,766 1,768 CD3-VH-G8 1,770 1,772 1,774 1,776 CD3-VH-G9 1,778 1,780 1,782 1,784 CD3-VH-G10 1,786 1,788 1,790 1,792 CD3-VH-G11 1,794 1,796 1,798 1800 CD3-VH-G12 1802 1804 1806 1808 CD3-VH-G13 1810 1812 1814 1816 CD3-VH-G14 1818 1820 1822 1824 CD3-VH-G15 1826 1828 1830 1832 CD3-VH-G16 1834 1836 1838 1840 CD3-VH-G17 1842 1844 1846 1848 CD3-VH-G18 1850 1852 1854 1856 CD3-VH-G19 1858 1860 1862 1864 CD3-VH-G20 1866 1868 1870 1872 CD3-VH-G21 1874 1876 1878 1880 CD3-VH-P 1882 1884 1886 1888

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Table 23

Heavy chain nucleic acid sequence identifiers

Designation CD3-VH Antibodies SEQ ID NO: HCVR CDR1 CDR2 CDR3 CD3-VH-G 1,729 1,731 1,733 1,735 CD3-VH-G2 1737 1,739 1,741 1,743 CD3-VH-G3 1,745 1747 1,749 1,751 CD3-VH-G4 1,753 1,755 1757 1,759 CD3-VH-G5 1761 1763 1765 1767 CD3-VH-G8 1,769 1,771 1,773 1,775 CD3-VH-G9 1,777 1,779 1,781 1,783 CD3-VH-G10 1785 1787 1789 1791 CD3-VH-G11 1,793 1,795 1,797 1,799 CD3-VH-G12 1801 1803 1805 1807 CD3-VH-G13 1809 1811 1813 1815 CD3-VH-G14 1817 1819 1821 1823 CD3-VH-G15 1825 182 7 1829 1831 CD3-VH-G16 1833 1835 1837 1839 CD3-VH-G17 1841 1843 1845 1847 CD3-VH-G18 1849 1851 1853 1855 CD3-VH-G19 1857 1859 1861 1863 CD3-VH-G20 1865 1867 1869 1871 CD3-VH-G21 1873 1875 1877 1879 CD3-VH-P 1881 1883 1885 1887

Table 24

Light chain amino acid sequence identifiers

Designation Antibodies SEQ ID NO: LCVR CDR1 CDR2 CDR3 VK1-39JK5 1890 1892 1894 1896

Table 25

Light chain nucleic acid sequence identifiers

Designation Antibodies SEQ ID NO: LCVR CDR1 CDR2 CDR3 VK1-39JK5 1889 1891 1893 1895

Antibody control 1, designated CD3-L2K, was constructed based on a known anti-CD3 antibody (ie anti-CD3 antibody L2K, as described in WO 2004/106380).

The isotype control antibody referred to in the examples herein is an isotype-matched (modified IgG4) antibody that reacts with a foreign antigen, i.e. FelD1 antigen.

Example 15. In vitro and in vivo studies on human monoclonal anti-CD3 antibodies.

In vivo and in vitro studies of human monoclonal anti-CD3 antibodies were carried out as described in US publication 2014/0088295, published March 27, 2014, and PCT/US2016/044732, filed July 29, 2016, which are hereby included in as a reference.

Certain human monoclonal anti-CD3 antibodies of the present invention bind soluble heterodimeric CD3 protein, either in antibody-capture or antigen-capture formats, with high affinity. Soluble heterodimeric CD3 protein (hCD3-epsilon/hCD3-delta; SEQ ID NO: 1900/1901) was produced with either a human Fc tag (hFcΔAdp/hFc; SEQ ID NO: 1931/1932) or a mouse Fc tag (mFcΔAdp/mfc ; SEQ ID NO: 1933/1934). CD3 heterodimeric protein was purified using

- 82 045730 the method described in Davis et al. (US2010/0331527).

Certain human monoclonal anti-CD3 antibodies of the present invention are bound to human T cells and induce T cell proliferation. Certain human monoclonal anti-CD3 antibodies of the present invention bind to monkey CD2+CD4+ T cells and induce their proliferation. Several human monoclonal anti-CD3 antibodies supported redirected T cell-mediated killing through Fc/FcR interaction in a calcein-based U937 killing assay. The observed killing, which is believed to depend on the interaction of the Fc antibody with the Fc receptor on U937 cells leading to clustering of CD3 on adjacent T cells, was suppressed by the addition of nonspecific human IgG (data not shown). A wide range of bispecific antibodies are activated using the anti-CD3 arm variants described herein (specifically, anti-CD3 arms based on the CD3-VH-P heavy chain derived from IGHV3-9*01, IGHJ6*02, IGHD5-12*01) human PBMC and monkey PBMC, and exhibit cytotoxic activity in cell lines expressing tumor antigen.

Example 16 Screening and identification of monoclonal anti-MUC16 antibodies suitable for immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded specimens.

Human tumor cell lines with known levels of MUC16 expression were identified, fixed in 10% neutral buffered formalin, and embedded in paraffin. These lines were used to screen various anti-MUC16 antibodies to identify candidates for IHC studies.

Cell lines include the following MUC16.

Negative cell lines: HT29 (colon) and PC3/ATCC parent (prostate); pancreatic cell lines with low or zero levels of MUC16: Capanl (pancreatic adenocarcinoma), HPAC (pancreatic adenocarcinoma). Endogenously MUC16-expressing cell lines include: OVCAR3 (ovarian) and PEO-1 (serous ovarian cancer).

Transfected cell lines were constructed as follows: PC3/ATCC (parental prostate cancer line) cells were transfected to generate PC3/MUC16 short and PC3/MUC16 tall cell lines. Both constructs contain the C-terminal MUC16 domain of amino acids 13810-14507 (from SEQ ID NO: 1899), and this contains part of the SEA12, SEA13, SEA14, SEA15, SEA16 domain, the C-terminal non-SEA region, the transmembrane region and the cytoplasmic domain . In addition, cells with high levels of PC3/MUC have additional N-terminal amino acids 12783-13467 (from SEQ ID NO: 1899), which include SEA5 (partial) through SEA9 and a short linker between the SEA9 domain and the start of the short MUC16 construct. This allows the differentiation of anti-MUC16 antibodies that bind to the repeat region from those that bind to the nub portion of MUC16 adjacent to the membrane after enzymatic cleavage and release of the repeat regions (similar to the CA125 portion of MUC16).

All staining was performed on a Ventana Discovery XT autostainer using standard protocols. The cell pellet was deparaffinized, heat-induced epitope retrieval was optimized, endogenous biotin was blocked, and protein blocking was performed. Antibodies were applied manually at an initial concentration of 10 μg/ml and also titrated to ensure signal specificity. Comparisons were made with a commercially available anti-MUC16 antibody (OC-125), which is specific for the CA-125 repeat regions (Roche, Ventana Catalog# 7602610), and with a negative control (no primary antibody). Detection was performed using donkey anti-mouse biotin followed by streptavidin-horseradish peroxidase. Conversion of the substrate, diamino-benzidine (DAB), was observed as a brown color. Samples were counterstained with hematoxylin to visualize nuclei. The results of staining experiments are presented in table. 26 below.

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Table 26

Binding of anti-MiC16 antibodies to MiC16-negative cells and cells expressing MUC16 or membrane-proximal portions of MUC16

Cell line: NT29 Capanl NRAS OVCAR3 PEO1 RSZ/ ATCC parent SKY RSZ/ MUC16 short RSZ/ MUC16 high H1M7130N - - - +++ ++ - +++ +++ H2aM7128N - - + +++ ++ - +++ +++ H2aM7131N - - - +++ ++ - +++ +++ H2aM7133N - - - +++ ++ - - - H2aM7138N - - + +++ ++ - +++ +++ H1M9519N - NT NT +++ +++ NT - +++ H3M9525N - NT NT - - NT - - OS-125 - - + +++ ++ - - +++ Negative control - - - - - - - -

NT - not tested.

Four of the antibodies tested (H1M7130N, H2aM7128N, H2aM7131N and H2aM7138N) showed positive binding to cells expressing the nub portion of MUC16 without repeat regions (PC3/MUC16 short). These antibodies have been identified as nub binders. One of the antibodies tested (H1M9519N) showed positive binding to cells expressing MUC16 repeat regions (PC3/MUC16 high), but negative binding to cells expressing only the nub portion of MUC16 (PC3/MUC16 short). This antibody is identified as a repeat binder similar to the commercially available antibody OC-125. The test antibodies are expected to bind tissues or cells of various origins having expressed proteins and/or protein fragments as described herein.

The present invention should not be limited in scope to the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Claims (27)

1. An isolated antibody or antigen-binding fragment thereof that binds to one or more of the five membrane-proximal domains of human MUC 16 SEA (SEQ ID NO: 1899), wherein the isolated antibody or antigen-binding fragment thereof binds within residues 14237-14290 of SEQ ID NO: 1899 and contains the complementarity determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 containing the amino acid sequences of SEQ ID NOs: 20, 22, 24, 28, 30 and 32, respectively. 2. An isolated antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 18, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 26. 3. An antibody-drug conjugate (ADC) for the treatment of MUC16-expressing cancer, comprising an anti-MUC16 antibody or an antigen-binding fragment thereof according to claim 1 or 2 and a cytotoxic agent, wherein the antibody or an antigen-binding fragment thereof and the cytotoxic agent are covalently linked through a linker. 4. The ADC according to claim 3, wherein the cytotoxic agent is selected from auristatin, maytansinoid, tubulisin, tomaimycin derivative or dolastatin derivative. 5. The ADC according to claim 3 or 4, wherein the cytotoxic agent is an auristatin selected from MMAE or MMAF, or a maytansinoid selected from DM1 or DM4. - 84 045730 6. ADC according to claim 3 or 4, wherein the cytotoxic agent is a maytansinoid having the structure O CH ₃ (Formula I), where A represents arylene or geoarylene; or (Formula II), where A _3a represents an amino acid, a peptide having 2-20 amino acids, alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, -CR5R6-, -O-, -C(=O)-, -О-С(=О)-, -С(=О)-О-, -OC(=O)-O-, -C(=O)-(CH _x )pi-, -С(=О) -O-(CHx)p1-, -(CH _x )p1-C(=O)-, -(CH _x )p _G C(=O)-O-, CHO-CCH^·, -((CH^ ^-, -C(=S)-, -C(=S)-S-, -C(=S)-NH-, -SC(=S)-, -SC(=S)-S- -S -, -so-, -SO2-, -NR ₄ -, -N(R ₄ )-C(=O)-N(R ₈ )-, -N(R ₄ )-C(=O)O-, -N(R ₄ )-C(=O)-, -C(=O)-N(R4)-, -C(=O)-N(R4)-C(=O)- or -OC(= O)-NR4-, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are optionally substituted, and p1, p2 and p3 are each independently 0 or an integer from 1 to 100; x represents 0, 1 or 2; R4, R5, R6 and _R8 are each independently H or substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl or heterocyclyl; And R4 _a is substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl or heterocyclyl. 7. ADC according to claim 6, wherein the maytansinoid is NON.R ^CH 3 9 ^NC O CH ₃ 8. ADC according to claim 6, wherein the maytansinoid is O CH ₃ - 85 045730 9. ADC according to claim 6, where the maytansinoid is 10. An ADC according to claim 3 or 4, containing an anti-MUC16 antibody or an antigen-binding fragment thereof and where * is the bond to the anti-MUC16 antibody or its antigen-binding fragment. 11. ADC according to claim 3 or 4, containing an anti-MUC16 antibody or an antigen-binding fragment thereof and where “ is the link to an anti-MUC16 antibody or an antigen-binding fragment thereof. 12. An ADC according to claim 3 or 4, containing an anti-MUC16 antibody or an antigen-binding fragment thereof and where ^r is a bond to an anti-MUC16 antibody or an antigen-binding fragment thereof. 13. ADC according to any one of claims 10-12, where the bond contacts the antibody or antigen-binding fragment through the sulfur component of the cysteine residue. - 86 045730 14. ADC according to claim 3 or 4, containing an anti-MUC16 antibody or an antigen-binding fragment thereof and o or o , or a mixture thereof, where it is a link to an anti-MUC16 antibody or an antigen-binding fragment thereof. 15. The ADC of claim 14, wherein the bond contacts the antibody or antigen-binding fragment thereof through the nitrogen moiety of the lysine residue. 16. ADC according to any one of claims 3 to 15, wherein the ADC contains from 1 to 4 cytotoxic agents per antiMUC16 antibody or antigen binding fragment thereof. 17. A method for detecting MUC16 in a biological sample that has been obtained from a subject, comprising detecting the presence of MUC16 in the biological sample by contacting said biological sample with an antibody or antigen binding fragment thereof according to claim 1 or 2 and detecting binding between MUC16 and the antibody or antigen binding fragment, where the biological sample is a tissue sample or a fluid sample. 18. The method according to claim 17, in which the tissue sample or fluid sample is selected from blood plasma, blood serum, ascites, ovary, uterus, cervix, liver, bladder, pancreas, stomach, small or large intestine, gall bladder, breast, lung, kidney, salivary and lacrimal glands or any of their malignant epithelioid formations. 19. A nucleic acid molecule containing a nucleotide sequence encoding an antibody or antigen-binding fragment according to claim 1 or 2. 20. A combination of nucleic acid molecules containing nucleotide sequences respectively encoding the variable region of the heavy chain of an antibody or antigen-binding fragment, and the variable region of the light chain of an antibody or antigen-binding fragment according to claim 1 or 2. 21. An expression vector containing a nucleic acid molecule according to claim 19. 22. A combination of expression vectors, respectively containing a combination of nucleic acid molecules according to claim 20. 23. A host cell comprising an antibody or antigen-binding fragment as claimed in claim 1 or 2, a nucleic acid molecule as claimed in claim 19, a combination of nucleic acid molecules as claimed in claim 20, an expression vector as claimed in claim 21, or a combination of expression vectors as claimed in claim 22. 24. A method for producing an anti-MUC16 antibody or an antigen-binding fragment thereof, comprising culturing a host cell according to claim 23 under conditions allowing the production of the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment. 25. The method of claim 24, wherein the host cell is a Chinese hamster ovary cell. 26. The method according to claim 24 or 25, further comprising providing a composition as a pharmaceutical composition containing an antibody or antigen-binding fragment and a suitable carrier, wherein the pharmaceutical composition is used for treating MUC16-expressing cancer. 27. Ahtu-MUC16 antibody or antigen-binding fragment thereof, which can be obtained by the method according to claim 24 or 25.